The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru.

Background: Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied. Results: We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits. Conclusions: As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.

Since the onset of the pandemic, many SARS-CoV-2 variants have emerged, exhibiting substantial evolution in the virus’ spike protein¹, the main target of neutralizing antibodies². A plausible hypothesis proposes that the virus evolves to evade antibody-mediated neutralization (vaccine- or infection-induced) to maximize its ability to infect an immunologically experienced population^1,3. Because viral infection induces neutralizing antibodies, viral evolution may thus navigate on a dynamic immune landscape that is shaped by local infection history. Here we developed a comprehensive mechanistic model, incorporating deep mutational scanning data^4,5, antibody pharmacokinetics and regional genomic surveillance data, to predict the variant-specific relative number of susceptible individuals over time. We show that this quantity precisely matched historical variant dynamics, predicted future variant dynamics and explained global differences in variant dynamics. Our work strongly suggests that the ongoing pandemic continues to shape variant-specific population immunity, which determines a variant’s ability to transmit, thus defining variant fitness. The model can be applied to any region by utilizing local genomic surveillance data, allows contextualizing risk assessment of variants and provides information for vaccine design.

Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. Here, we use developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, we show that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors.

Antiviral pre-exposure prophylaxis (PrEP) through daily drug administration can protect healthy individuals from HIV-1 infection. While PrEP was recently approved by the FDA, the potential long-term consequences of PrEP implementation remain entirely unclear. The aim of this study is to predict the efficacy of different prophylactic strategies with the pro-drug tenofovir-disoproxil-fumarate (TDF) and to assess the sensitivity towards timing- and mode of TDF administration (daily- vs. single dose), adherence and the number of transmitted viruses. We developed a pharmacokinetic model for TDF and its active anabolite tenofovir-diphosphate (TFV-DP) and validated it with data from 4 different trials, including 4 distinct dosing regimes. Pharmacokinetics were coupled to an HIV model and viral decay following TDF mono-therapy was predicted, consistent with available data. Subsequently, a stochastic approach was used to estimate the % infections prevented by (i) daily TDF-based PrEP, (ii) one week TDF started either shortly before, or -after viral exposure and (iii) a single dose oral TDF before viral challenge (sd-PrEP). Analytical solutions were derived to assess the relation between intracellular TFV-DP concentrations and prophylactic efficacy. The predicted efficacy of TDF was limited by a slow accumulation of active compound (TFV-DP) and variable TFV-DP half-life and decreased with increasing numbers of transmitted viruses. Once daily TDF-based PrEP yielded 80% protection, if at least 40% of pills were taken. Sd-PrEP with 300 mg or 600 mg TDF could prevent 50% infections, when given at least before virus exposure. The efficacy dropped to 10%, when given 1 h before 24 h exposure. Efficacy could not be increased with increasing dosage or prolonged administration. Post-exposure prophylaxis poorly prevented infection. The use of drugs that accumulate more rapidly, or local application of tenofovir gel may overcome the need for drug administration long before virus exposure.