ProBac-seq, a bacterial single-cell RNA sequencing methodology using droplet microfluidics and large oligonucleotide probe sets.

Methods that measure the transcriptomic state of thousands of individual cells have transformed our understanding of cellular heterogeneity in eukaryotic cells since their introduction in the past decade. While simple and accessible protocols and commercial products are now available for the processing of mammalian cells, these existing technologies are incompatible with use in bacterial samples for several fundamental reasons including the absence of polyadenylation on bacterial messenger RNA, the instability of bacterial transcripts and the incompatibility of bacterial cell morphology with existing methodologies. Recently, we developed ProBac sequencing (ProBac-seq), a method that overcomes these technical difficulties and provides high-quality single-cell gene expression data from thousands of bacterial cells by using messenger RNA-specific probes. Here we provide details for designing large oligonucleotide probe sets for an organism of choice, amplifying probe sets to produce sufficient quantities for repeated experiments, adding unique molecular indexes and poly-A tails to produce finalized probes, in situ probe hybridization and single-cell encapsulation and library preparation. This protocol, from the probe amplification to the library preparation, requires ~7 d to complete. ProBac-seq offers several advantages over other methods by capturing only the desired target sequences and avoiding nondesired transcripts, such as highly abundant ribosomal RNA, thus enriching for signal that better informs on cellular state. The use of multiple probes per gene can detect meaningful single-cell signals from cells expressing transcripts to a lesser degree or those grown in minimal media and other environmentally relevant conditions in which cells are less active. ProBac-seq is also compatible with other organisms that can be profiled by in situ hybridization techniques.

Efficient prime editing in two-cell mouse embryos using PEmbryo.

Using transient inhibition of DNA mismatch repair during a permissive stage of development, we demonstrate highly efficient prime editing of mouse embryos with few unwanted, local byproducts (average 58% precise edit frequency, 0.5% on-target error frequency across 13 substitution edits at 8 sites), enabling same-generation phenotyping of founders. Whole-genome sequencing reveals that mismatch repair inhibition increases off-target indels at low-complexity regions in the genome without any obvious phenotype in mice.

Probe-based bacterial single-cell RNA sequencing predicts toxin regulation.

Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. Understanding all cell states requires studying isogenic bacterial populations at the single-cell level. Here we developed probe-based bacterial sequencing (ProBac-seq), a method that uses libraries of DNA probes and an existing commercial microfluidic platform to conduct bacterial single-cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. Applied to Bacillus subtilis and Escherichia coli, ProBac-seq correctly identifies known cell states and uncovers previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, application of the approach to Clostridium perfringens reveals heterogeneous expression of toxin by a subpopulation that can be controlled by acetate, a short-chain fatty acid highly prevalent in the gut. Overall, ProBac-seq can be used to uncover heterogeneity in isogenic microbial populations and identify perturbations that affect pathogenicity.

Novel radiation therapy approaches for breast cancer treatment.

The role of radiation therapy in the management of breast cancer continues to evolve. For patients with early stage breast cancer, hypofractionated whole breast irradiation following breast conserving surgery now represents the standard of care based on randomized data with long-term efficacy and toxicity outcomes. Partial breast irradiation has been found, in several randomized trials, to be effective and appropriate in selected patients with the potential to reduce toxicities as compared to whole breast irradiation. The study of tumor biology and genetics and its role in radiation therapy decision making continues to grow and the advances may help identify patients where radiation therapy can be safely omitted, with future studies looking at de-intensification approaches. Recent randomized data has demonstrated a growing role for regional nodal irradiation in patients with more advanced disease, with future studies looking to identify whether nodal radiation is indicated following neoadjuvant chemotherapy or with certain favorable tumor biologies. While postmastectomy radiation therapy represents a standard approach for patients with locally advanced breast cancer, new data supports the role of hypofractionated regimens as well as its use in patients previously considered lower risk with unfavorable tumor biology. Oligometastatic disease represents a new area of study in breast cancer with prospective trials underway and current data supporting consideration of techniques such as stereotactic body radiation therapy in appropriately selected patients.

An agent-based model to investigate microbial initiation of Alzheimer's via the olfactory system.

BACKGROUND: Alzheimer's disease (AD) is a degenerative brain disease. A novel agent-based modelling framework was developed in NetLogo 3D to provide fundamental insights into the potential mechanisms by which a microbe (eg. Chlamydia pneumoniae) may play a role in late-onset AD. The objective of our initial model is to simulate one possible spatial and temporal pathway of bacterial propagation via the olfactory system, which may then lead to AD symptoms. The model maps the bacteria infecting cells from the nasal cavity and the olfactory epithelium, through the olfactory bulb and into the olfactory cortex and hippocampus regions of the brain. RESULTS: Based on the set of biological rules, simulated randomized infection by the microbe led to the formation of beta-amyloid (Aß) plaque and neurofibrillary (NF) tangles as well as caused immune responses. Our initial simulations demonstrated that breathing in C. pneumoniae can result in infection propagation and significant buildup of Aß plaque and NF tangles in the olfactory cortex and hippocampus. Our model also indicated how mucosal and neural immunity can play a significant role in the pathway considered. Lower immunities, correlated with elderly individuals, had quicker and more Aß plaque and NF tangle formation counts. In contrast, higher immunities, correlated with younger individuals, demonstrated little to no such formation. CONCLUSION: The modelling framework provides an organized visual representation of how AD progression may occur via the olfactory system to better understand disease pathogenesis. The model confirms current conclusions in available research but can be easily adjusted to match future evidence and be used by researchers for their own individual purposes. The goal of our initial model is to ultimately guide further hypothesis refinement and experimental testing to better understand the dynamic system interactions present in the etiology and pathogenesis of AD.