Single Replication Research Articles

A simple phylogenetic approach to analyze hypermutated HIV proviruses reveals insights into their dynamics and persistence during antiretroviral therapy

Abstract Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, hypermutated sequences are routinely excluded from phylogenetic trees because their extensive mutations complicate phylogenetic inference, and as a result we know relatively little about their within-host evolutionary origins and dynamics. Using >1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up − including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and >500 proviruses isolated over a median 9 years on ART − we evaluated three approaches for masking hypermutation in nucleotide alignments. Our goals were to 1) reconstruct phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env-intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env-intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, supporting the use of these corrected trees for molecular dating. Subsequent molecular dating of hypermutated proviruses revealed that these sequences spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env-intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus’ evolutionary histories reveal insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

Non-Linear Saturated Multi-Objective Pseudo-Screening Using Support Vector Machine Learning, Pareto Front, and Belief Functions: Improving Wastewater Recycling Quality

Increasing wastewater treatment efficiency is a primary aim in the circular economy. Wastewater physicochemical and biochemical processes are quite complex, often requiring a combination of statistical and machine learning tools to empirically model them. Since wastewater treatment plants are large-scale operations, the limited opportunities for extensive experimentation may be offset by miniaturizing experimental schemes through the use of fractional factorial designs (FFDs). A recycling quality improvement study that relies on non-linear multi-objective multi-parameter FFD (NMMFFD) datasets was reanalyzed. A published NMMFFD ultrafiltration screening/optimization case study was re-examined regarding how four controlling factors affected three paper mill recycling characteristic responses using a combination of statistical and machine learning methods. Comparative machine learning screening predictions were provided by (1) quadratic support vector regression and (2) optimizable support vector regression, in contrast to quadratic linear regression. NMMFFD optimization was performed by employing Pareto fronts. Pseudo-screening was applied by decomposing the replicated NMMFFD dataset to single replicates and then testing their replicate repeatability by introducing belief functions that sought to maximize credibility and plausibility estimates. Various versions of belief functions were considered, since the novel role of the three process characteristics, as independent sources, created a high level of conflict during the information fusion phase, due to the inherent divergent belief structures. Correlations between two characteristics, but with opposite goals, may also have contributed to the source conflict. The active effects for the NMMFFD dataset were found to be the transmembrane pressure and the molecular weight cut-off. The modified adjustment was pinpointed to the molecular weight cut-off at 50 kDa, while the optimal transmembrane pressure setting persisted at 2.0 bar. This mixed-methods approach may provide additional confidence in determining improved recycling process adjustments. It would be interesting to implement this approach in polyfactorial wastewater screenings with a greater number of process characteristics.

Intranasal M2SR and BM2SR Vaccine Viruses Do Not Shed or Transmit in Ferrets

Background/Objectives: Live influenza vaccines are considered to stimulate better overall immune responses but are associated with safety concerns regarding shedding and the potential for transmission or reassortment with wild-type influenza viruses. Intranasal M2SR and BM2SR (M2- and BM2-deficient single replication), intranasal influenza viruses, have shown promise as broadly cross-reactive next-generation influenza vaccines. The replication deficiency, shedding, and transmissibility of M2SR/BM2SR viruses were evaluated in a ferret model. Methods: Wild-type influenza A and B control viruses replicated in upper respiratory organs and transmitted to both direct and aerosol contact ferrets, whereas M2SR and BM2SR influenza vaccine viruses were not detected in any tissues or in nasal washes after inoculation and were not recovered from any direct or aerosol contact ferrets. Mice were simultaneously infected with wild-type influenza A and M2SR viruses to assess reassortment potential. Sequence and PCR analyses of the genome recovered from individual virus plaques isolated from lung homogenates identified the origin of the segments as exclusively from the replicating wild-type virus. Results: These results indicate that M2SR and BM2SR influenza vaccine viruses are attenuated, do not shed or transmit, and have a low probability for reassortment after coinfection. Absence of shedding was further demonstrated in nasal swabs taken from subjects who were inoculated with H3N2 M2SR in a previously described Phase 1 clinical study. Conclusions: These results indicate that M2SR/BM2SR viruses have the potential to be used in a broader population range than current live influenza vaccines.

A-101 Is That Unexpected? Education on Handling Real World Complications with Tightly Controlled Analytes During Method Evaluation

Abstract Background A common activity in clinical laboratories is performance evaluations. Given the importance for a new analyzer being put into clinical use, this exercise typically requires extensive preparation with significant time and resources invested to bring on new instrumentation. During early adoption, studies examined real-world performance and comparability of the Atellica® CI Analyzer (Atellica CI) to clinically used instruments. Precision, linearity, and method comparison (MC) studies, conducted in accordance with CLSI protocols, were completed at several locations installing new instruments to examine the analytical performance of common clinical laboratory assays. This work examines the impact of using only unmodified specimens to verify performance during method comparison for tightly controlled biological analytes like electrolytes and illustrates the impact of the tested sample range on regression analysis. Methods Testing was completed using unmodified, de-identified, remnant patient serum specimens. The MC studies for these electrolytes were completed over multiple days using single replicates from at least 40 individual specimens, on two instruments, the Atellica CI and the Dimension® EXL in accordance with CLSI EP09-3A. The effect of sample range on regression was demonstrated through data restriction and inclusion of modified sample results within the data analysis. Data analysis was completed using Analyse-it for Microsoft Excel (version 6.15.4). Results Representative method comparison data for sodium, chloride, and potassium found slope differences were more pronounced when sample concentrations were more limited. Individual assay slopes were determined using Deming linear regression model and comparisons between restricted samples show the effects of sample range on regression and correlation parameters. Conclusions Understanding the relationship between sample range and regression analysis is helpful to illustrate an important fundamental concept of method comparison. These examples illustrate the importance in covering as much of the analytical measuring interval as possible and the impact on regression analysis when samples are narrowly grouped.

Evaluation of INM Practices on Fruit Yield and Quality of Established Mango Orchard

The current study was conducted by Krishi Vigyan Kendra, Saharsa, under the supervision of Bihar Agricultural University, Sabour, Bhagalpur, Bihar, and ICAR-ATARI (Zone-IV) in fifteen farmers' fields in Bihar's Saharsa district between 2020-21 and 2021-22. Each farmer's field was handled as a single replication. The experimental treatments were distributed in a Randomized Block Design with 15 replications (=15 farmers) and three treatments containing recommended agronomic techniques. According to two years' worth of pooled data, treatment T3 (75% RDF + 20 kg Vermicompost + 250 ml Azotobacter + 250 ml PSB) produced the highest fruit length (9.35 cm), fruit width (6.52 cm), fruit volume (213.21 cm3), and fruit weight (225.35 g), as well as yield parameters, such as the highest number of fruits per plant (365.33), fruit yield per plant (91.33 kg), and TSS (18.10°Brix). in this respect, T2 (75% RDF + 20 kg Vermicompost + 250 ml Azotobacter) came to the next. Considering the effect of agronomic practices on some chemical properties of soil under trees, T3 (75% RDF + 20 kg Vermicompost + 250 ml Azotobacter + 250 ml PSB per tree) had the highest recorded values of pH (7.4), EC (0.51 dSm-1), Organic Carbon (0.52%), available N (305.21 kg/ha), available P (32.56 kg/ha), and available K (201.33 kg/ha). Also, the maximum soil microbial count of 6.5 × 109 and 7.8 × 109, was recorded. From an economical view point, treatment T3 resulted in the maximum benefit cost ratio (3.01) and the highest net realization (₹.497800) based on fruit yield per hectare.

Intranasal M2SR (M2-deficient Single Replication) Influenza Vaccine Induces Broadly Reactive Mucosal Antibody Production in Adults.

Intranasal M2SR (M2-deficient Single Replication influenza virus) vaccine induces robust immune responses in animal models and human subjects. A high-throughput multiplexed platform was used to analyze hemagglutinin-specific mucosal antibody responses in adults after a single dose of H3N2 M2SR. Nasal swab specimens were analyzed for total and hemagglutinin-specific IgA. Significant, dose-dependent increases in mucosal antibody responses to vaccine-matched and drifted H3N2 hemagglutinin were observed in M2SR vaccinated subjects regardless of baseline serum and mucosal immune status. These data suggest that M2SR induces broadly cross-reactive mucosal immune responses which may provide better protection against drifted and newly emerging influenza strains.

APEAch: Automated Pipeline for End-to-End Analysis of Epigenomic and Transcriptomic Data.

With the advent of next-generation sequencing (NGS), experimental techniques that capture the biological significance of DNA loci or RNA molecules have emerged as fundamental tools for studying the epigenome and transcriptional regulation on a genome-wide scale. The volume of the generated data and the underlying complexity regarding their analysis highlight the need for robust and easy-to-use computational analytic methods that can streamline the process and provide valuable biological insights. Our solution, aPEAch, is an automated pipeline that facilitates the end-to-end analysis of both DNA- and RNA-sequencing assays, including small RNA sequencing, from assessing the quality of the input sample files to answering meaningful biological questions by exploiting the rich information embedded in biological data. Our method is implemented in Python, based on a modular approach that enables users to choose the path and extent of the analysis and the representations of the results. The pipeline can process samples with single or multiple replicates in batches, allowing the ease of use and reproducibility of the analysis across all samples. aPEAch provides a variety of sample metrics such as quality control reports, fragment size distribution plots, and all intermediate output files, enabling the pipeline to be re-executed with different parameters or algorithms, along with the publication-ready visualization of the results. Furthermore, aPEAch seamlessly incorporates advanced unsupervised learning analyses by automating clustering optimization and visualization, thus providing invaluable insight into the underlying biological mechanisms.

A simple phylogenetic approach to analyze hypermutated HIV proviruses reveals insights into their dynamics and persistence during antiretroviral therapy.

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using > 1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up - including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and > 500 proviruses isolated over a median 9 years on ART - we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env-intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env-intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env-intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

Shiba: A unified computational method for robust identification of differential RNA splicing across platforms.

Alternative pre-mRNA splicing (AS) is a fundamental regulatory process that generates transcript diversity and cell type variation. We developed Shiba, a robust method integrating transcript assembly, splicing event identification, read counting, and statistical analysis, to efficiently quantify exon splicing levels across various types of RNA-seq datasets. Compared to existing pipelines, Shiba excels in capturing both annotated and unannotated or cryptic differential splicing events with superior accuracy, sensitivity, and reproducibility. Furthermore, Shiba's unique consideration of junction read imbalance and exon-body read coverage reduces false positives, essential for downstream functional analyses. We have further developed scShiba for single-cell/nucleus (sc/sn) RNA-seq data, enabling the exploration of splicing variations in heterogeneous cell populations. Both simulated and real data demonstrate Shiba's robustness across multiple sample sizes, including n=1 datasets and individual cell clusters from scRNA-seq. Application of Shiba on single replicates of RNA-seq identified new AS-NMD targets, and scShiba on snRNA-seq revealed intricate temporal AS regulation in dopaminergic neurons. Both Shiba and scShiba are provided in Docker/Singularity containers and Snakemake pipeline, enhancing accessibility and reproducibility. The comprehensive capabilities of Shiba and scShiba allow systematic and robust quantification of alternative splicing events, laying a solid foundation for mechanistic exploration of functional complexity in RNA splicing.

Sister chromatid cohesion establishment during DNA replication termination.

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

Emerging and comparative genomic analysis of a novel plasmid carrying blaKPC-2 in Citrobacter freundii

Objective: To explore the drug resistance mechanism and gene structure characteristics of a carbapenemase-producing novel incompatibility group plasmid pNY2385-KPC from Citrobacter freundii. Methods: A multi-drug resistant strain was obtained from urine samples of patients with fever in the emergency ward of Li Huili Hospital, Ningbo Medical Center. Bacterial species was preliminary identified and finally confirmed by 16S rRNA gene amplification and the average nucleotide identity alignment, respectively. The minimum inhibitory concentrations of the antimicrobial agents were determined by VITEK 2 Compact System. The complete genome sequence was obtained by "third-generation" sequencing methods, and then detailed annotation of gene function and comparative genomic analysis of plasmid structure were carried out by BLASTP/BLASTN, RefSeq, ConservedDomains, ResFinder, Isfinder, etc. Results: The pNY2385-KPC carried by citrobacter freundii NY2385 belonged a novel incompatibility group, and contained blaKPC-2 and conjugative transfer (type Ⅳ secretory system, T4SS) genes, which could induce conjugative transfer. A total of 15 plasmids of the same type as pNY2385-KPC were retrieved by NCBI, which were from Citrobacter freundii, and the rest were from Serratia marcescens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Raoultella planticola and other bacteria, and were broad-host-range plasmids. The sequence comparative analysis of all 6 of the novel plasmid from Citrobacter freundii showed that the structure of the novel plasmid had certain conserved property, with Tn6296 variant structure carrying blaKPC-2, and plasmid pCF1807-3 had both repApNY2385-KPC and repAIncX8. Conclusion: The pNY2385-KPC type plasmids in Citrobacter freundii carried blaKPC-2 resistance gene, which were divided into two subtypes: repApNY2385-KPC single replicator and repApNY2385-KPC/repAIncX8 complex replicator, belonging to broad-host-range plasmids. And as a mobile genetic element, the plasmids promote the spread of blaKPC-2.

1155. Coadministration of Intranasal M2SR (M2-deficient Single Replication) Investigational Influenza Vaccine with Fluzone High Dose Induces Superior Immune Responses to Fluzone High Dose Alone in 65-85 Year Old Adults

Abstract Background Adults over age 65 years exhibit increased susceptibility to influenza viruses, accounting for 70-85% of annual influenza-related US fatalities. Licensed vaccines mainly induce serum humoral immunity and remain the primary method of influenza prevention, but vaccine effectiveness (VE) remains particularly low in older individuals (Fig 1). Stimulation of mucosal antibodies and T cells, immune effectors also associated with protection against influenza, are anticipated to enhance VE. Methods A randomized, double-blind, double-dummy, placebo-controlled Phase 1b study (NCT05163847) was conducted at 6 US study sites with the investigational intranasal M2SR (M2 deficient Single Replication) and Fluzone High Dose (HD) vaccine that contained HA & NA from A/Cambodia/e0826360/2020 (Cam2020). Adults aged 65-85 years (n = 305) received either one administration of Cam2020 M2SR alone, Cam2020 M2SR along with HD, HD alone, or placebo (Table 1). Demographics were comparable across cohorts (Table 2). Results Study vaccination was well-tolerated. Intranasal M2SR generated significantly increased serum HAI and NAI antibodies compared with placebo (Fig 2). Moreover, M2SR coadministered with HD induced HAI and NAI antibodies in significantly higher proportion of subjects than HD alone (Fig 2). M2SR alone and M2SR coadministered with HD elicited significant mucosal secretory IgA (sIgA) antibodies against vaccine antigen, Cam2020, and drifted H3N2 Darwin2021 strain (Fig 3). Similarly, T cells positive for IFNg and GrzB secretion were significantly induced by M2SR only or by M2SR coadministered with HD (Fig 4). HD alone did not induce mucosal antibodies or T cell responses (Figs 3 & 4). Conclusion Coadministration of intranasal M2SR with intramuscular Fluzone HD resulted in a multi-faceted immune response in older adults aged 65-85 years old with a higher proportion of responders across all immune measurements compared with either vaccine alone. Coadministration of the two vaccines resulted in significant rises in serum HAI and NAI titers compared to HD alone (Fig 5). These broad immune responses support the potential for the safe coadministration of M2SR with HD to enhance protection against influenza infection in this highly vulnerable age group. Disclosures Joseph Eiden, MD, PhD, FluGen, Inc.: Advisor/Consultant Bryan Murray, M.D., FluGen, Inc.: Advisor/Consultant Renee Herber, B.S., FluGen, Inc.: Salary Roger Aitchison, ScM, FluGen, Inc.: Advisor/Consultant David Marshall, B.S., FluGen, Inc.: salary Michael Moser, Ph.D., FluGen, Inc.: salary Pamuk Bilsel, Ph.D., FluGen, Inc.: salary

Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.

Quantitative Analysis of Cellular Morphology During In Vitro Decidualization.

Decidualization is a differentiation process involving shape reorganization from a fibroblast to an epithelioid-like appearance characteristic of endometrial stromal cells. For the study of in vitro decidualization, one needs to check that the cells have undergone this process effectively. Verification is usually done by analyzing the expression of decidual markers, but changes in morphology are a more comprehensive feature. However, morphological specificities (i.e., flatness) of endometrial cells prevent the use of existing automated tools. A simple and accurate methodology was developed to quantify the phenotypic changes that occur in an in vitro decidualization system. This approach analyzes cell circularity directly from light microscopy images to follow the effects of progesterone or progestin R5020 in combination with estradiol (E2) and cAMP in inducing the decidualization of human endometrial cells. A statistical model to detect the differences in the kinetics of decidualization of the two hormonal stimuli before all the cell population acquire the decidual phenotype was implemented. It was found that statistical differences in morphology between decidualized and control cells could be detected 2 days after the treatments. Here we detail the model applied, scripts, and input files in order to provide a useful, practical, and low-cost tool to evaluate morphological aspects of endometrial stromal differentiation. This method allows the verification of the effectiveness of the decidualization process of the stromal endometrial cells without having to use cell replicates, as other methods such as immunofluorescence and RT-qPCR assays require. Consequently, this approach can follow the kinetics of a living single replicate throughout the experiment. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell circularity quantification of human stromal endometrial cells using ImageJ Basic Protocol 2: Statistical analysis of cell circularity of human stromal endometrial cells.

B-097 Storage Stability of HbA1c in Whole Blood Tubes at Room Temperature on the Abbott Alinity c Clinical Chemistry System

Abstract Background Hospital laboratories frequently receive requests to order additional tests on an existing specimen. For this reason, laboratories routinely save specimens after initial testing has been completed for a defined period of time. The current literature lacks specific information regarding HbA1c stability in whole blood and the effects of red blood cell settling. In addition, literature references are not inclusive of various conditions which can cause gaps in data collection such as samples in queue for prolonged periods. To obtain information on prolonged storage of whole blood tubes, HbA1c was assessed for storage at room temperature (RT) (20–25°C) for up to 4 hours on the Alinity c system. In addition, potential carryover from a whole blood to a serum sample was assessed. Methods A minimum of 40 whole blood sample pools (approx. 8 mLs) were prepared from remnant samples. Sample pools were mixed, aliquoted, and stored at RT on the Alinity c system. The samples were tested as a single replicate continuously for up to 4 hours without mixing (minimum of 20 cycles) and compared to the baseline sample values. Carryover from a whole blood to a glucose serum sample was assessed after 4 hours continuous testing of whole blood to mimic a high volume testing laboratory. The stability of each HbA1c sample and carryover into a glucose sample were evaluated by comparing the mean absolute or percent difference from the respective sample baseline measurement. The allowed deviation from baseline was 5%. Results After 4 hours at RT, the whole blood cells had settled to approximately 50% from the top of the tube. The red blood cell settling had no significant impact on the results. Within sample precision for the 20 cycles of repetitive testing was </ = 1%. The 48 individual samples showed insignificant difference from the baseline value, with a mean change of 1.0% (Range 0–3.2%). Sample carryover from the whole blood samples to a serum glucose sample was not significant (2.71 Baseline vs 2.64 first replicate). Conclusion This study demonstrated that the storage stability of HbA1c is stable for up to 4 hours at RT on the Alinity c system.

A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells.

When replication forks encounter DNA lesions that cause polymerase stalling, a checkpoint pathway is activated. The ATR-dependent intra-S checkpoint pathway mediates detection and processing of sites of replication fork stalling to maintain genomic integrity. Several factors involved in the global checkpoint pathway have been identified, but the response to a single replication fork barrier (RFB) is poorly understood. We utilized the Escherichia coli-based Tus-Ter system in human MCF7 cells and showed that the Tus protein binding to TerB sequences creates an efficient site-specific RFB. The single fork RFB was sufficient to activate a local, but not global, ATR-dependent checkpoint response that leads to phosphorylation and accumulation of DNA damage sensor protein γH2AX, confined locally to within a kilobase of the site of stalling. These data support a model of local management of fork stalling, which allows global replication at sites other than the RFB to continue to progress without delay.

Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Improving Experimental Design through Uncertainty Analysis

In this paper, the development of a fission-gas collecting and physical-analysis-enabling instrument was proposed for small-volume determination. Analysis specifications require a design capable of accurately and repeatably determining volumes in the range of 0.07–2.5 mL. This system relies on a series of gas expansions originating from a cylinder with known internal volume. The combined gas law is used to derive the unknown volumes from these expansions. Initial system designs included one of two known volumes, 11.85 ± 0.34 mL and 5.807 ± 0.078 mL, with a manifold volume of 32 mL. Results obtained from modeling this system’s operation showed that 0.07 mL can be determined with a relative expanded uncertainty greater than 300% (k = 2) for a single replicate, which was unacceptable for the proposed experimental design. Initial modeling showed that the volume connecting the known volume and rodlet, i.e., the manifold volume, and the sensitivity of the pressure sensor were key contributors to the expanded uncertainty of the measured rodlet volume. The system’s design limited the available options for pressure sensors, so emphasis was placed on the design of the manifold volume. The final system design reduced the manifold volume to 17 mL. These changes in design, combined with replicate analysis, were able to reduce the relative expanded uncertainty by ±12% (k = 2) for the 0.07 mL volume.

Intranasal Single-Replication Influenza Vector Induces Cross-Reactive Serum and Mucosal Antibodies against SARS-CoV-2 Variants.

Current SARS-CoV-2 vaccines provide protection for COVID-19-associated hospitalization and death, but remain inefficient at inhibiting initial infection and transmission. Despite updated booster formulations, breakthrough infections and reinfections from emerging SARS-CoV-2 variants are common. Intranasal vaccination to elicit mucosal immunity at the site of infection can improve the performance of respiratory virus vaccines. We developed SARS-CoV-2 M2SR, a dual SARS-CoV-2 and influenza vaccine candidate, employing our live intranasal M2-deficient single replication (M2SR) influenza vector expressing the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein of the prototype strain, first reported in January 2020. The intranasal vaccination of mice with this dual vaccine elicits both high serum IgG and mucosal IgA titers to RBD. Sera from inoculated mice show that vaccinated mice develop neutralizing SARS-CoV-2 antibody titers against the prototype and Delta virus strains, which are considered to be sufficient to protect against viral infection. Moreover, SARS-CoV-2 M2SR elicited cross-reactive serum and mucosal antibodies to the Omicron BA.4/BA.5 variant. The SARS-CoV-2 M2SR vaccine also maintained strong immune responses to influenza A with high titers of anti H3 serum IgG and hemagglutination inhibition (HAI) antibody titers corresponding to those seen from the control M2SR vector alone. With a proven safety record and robust immunological profile in humans that includes mucosal immunity, the M2SR influenza viral vector expressing key SARS-CoV-2 antigens could provide more efficient protection against influenza and SARS-CoV-2 variants.

Asphalt mixture fatigue damage and failure predictions using the simplified viscoelastic continuum damage (S-VECD) model

Fatigue cracking is a primary asphalt pavement distress and various models have been developed to predict the fatigue life of asphalt mixtures using laboratory tests. One such model is the simplified viscoelastic continuum damage (S-VECD) model, which has been implemented in the pavement performance prediction program, FlexPAVETM. The S-VECD model test protocols (AASHTO TP 133 and AASHTO T 400) and data processing tool (FlexMATTM) are widely used around the world. Over the past three decades, this model has been continuously improved and refined. However, questions remain on the model’s ability to predict the material response when stress or strain are used as the model input. Also, different fitting procedures for the model calibration were found to affect the model’s prediction accuracy. In this study, analysis was conducted using data for four typical North Carolina mixes based on single replicate tests and multiple replicate tests to compare the model’s prediction accuracy based on stress versus strain as the model input and by considering fitting errors in the different calculations. The results show that using strain as the model input, which automatically incorporates portions of permanent strain, yields more accurate predictions compared to using stress as the input, regardless of the fitting algorithm. Additionally, in the analysis of individual test data, which is not affected by replicate specimen variability, the model’s predictions match the measured data well, as long as the fitting errors of the damage characteristic curve are controlled. When the data from replicate tests are analyzed together, although specimen variability compromises the S-VECD model’s prediction accuracy, failure can still be reasonably determined when strain is used as the model input.