Morbidity and mortality of Serratia marcescens bacteraemia during the substance use epidemic.

BACKGROUND: Serratia marcescens (S. marcescens) is an Enterobacterales species present throughout the environment and causes a range of infections. Historically, S. marcescens has been associated with persons who inject drugs (PWID), but literature is scarce. This study aimed to compare treatment characteristics and clinical outcomes between PWID and non-PWID with Serratia marcescens bacteraemia. METHODS: This was a retrospective cohort study of patients hospitalised with S. marcescens bacteraemia from 1 January 2013 to 31 December 2019 at a tertiary medical centre. Patients were included if they were aged ≥ 18 years and had at least one positive blood culture for S. marcescens. RESULTS: Of the 67 patients who met inclusion criteria, 14 were identified as PWID (21%) and 53 were non-PWID (79%). Persons who inject drugs were younger (median age: PWID 32 years, non-PWID 67 years) and less likely to have renal disease (PWID 7%, non-PWID 34%). Persons who inject drugs had a higher incidence of infective endocarditis (IE) (PWID 48%, non-PWID 0%) and were more likely to receive combination antimicrobial therapy (PWID 29%, non-PWID 2%). All-cause mortality at 12 months was comparable between groups (PWID 21%, non-PWID 21%). CONCLUSION: This study demonstrates that long-term outcomes of PWID are comparable with non-PWID, despite PWID being a younger cohort with fewer comorbidities. Clinicians should have high suspicion of IE in PWID with S. marcescens bacteraemia.

Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer.

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2-7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

Synthetic feedback control using an RNAi-based gene-regulatory device.

BACKGROUND: Homeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response. The development of biological components that can respond to changes in the cellular environment and interface with endogenous molecules would enable more sophisticated genetic circuits and greatly advance our cellular engineering capabilities. RESULTS: Here we describe a platform that combines a ligand-responsive ribozyme switch and synthetic miRNA regulators to create an OFF genetic control device based on RNA interference (RNAi). We developed a mathematical model to highlight important design parameters in programming the quantitative performance of RNAi-based OFF control devices. By modifying the ribozyme switch integrated into the system, we demonstrated RNAi-based OFF control devices that respond to small molecule and protein ligands, including the oncogenic protein E2F1. We utilized the OFF control device platform to build a negative feedback control system that acts as a proportional controller and maintains target intracellular protein levels in response to increases in transcription rate. CONCLUSIONS: Our work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device. The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands. Finally, the RNAi-based OFF switch can be used to implement a negative feedback control system, which maintains target protein levels around a set point level. The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.

A quantitative framework for the forward design of synthetic miRNA circuits.

Synthetic genetic circuits incorporating regulatory components based on RNA interference (RNAi) have been used in a variety of systems. A comprehensive understanding of the parameters that determine the relationship between microRNA (miRNA) and target expression levels is lacking. We describe a quantitative framework supporting the forward engineering of gene circuits that incorporate RNAi-based regulatory components in mammalian cells. We developed a model that captures the quantitative relationship between miRNA and target gene expression levels as a function of parameters, including mRNA half-life and miRNA target-site number. We extended the model to synthetic circuits that incorporate protein-responsive miRNA switches and designed an optimized miRNA-based protein concentration detector circuit that noninvasively measures small changes in the nuclear concentration of ß-catenin owing to induction of the Wnt signaling pathway. Our results highlight the importance of methods for guiding the quantitative design of genetic circuits to achieve robust, reliable and predictable behaviors in mammalian cells.

Construction of ligand-responsive microRNAs that operate through inhibition of Drosha processing.

MicroRNAs (miRNAs) offer powerful tools for targeted gene silencing in almost all eukaryotes. These tools have received considerable attention for their utility in both fundamental genetic studies and as therapeutic agents. Rendering individual microRNAs responsive to endogenous or exogenously applied molecules (or ligands) can improve the stringency of silencing and can mediate autonomous control. This chapter describes the construction of ligand-responsive miRNAs that undergo reduced processing and subsequent gene silencing when bound by the recognized ligand. Following a simple set of rules, the engineered microRNAs can be readily modified to target different sequences and to bind different ligands. Individual miRNAs also can be incorporated into the same transcript for tunable, multi-gene silencing.

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration.

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles - the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions.

Engineering biological systems with synthetic RNA molecules.

RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors.

The perinuclear actin cap in health and disease.

We recently demonstrated the existence of a previously uncharacterized subset of actomyosin fibers that form the perinuclear actin cap, a cytoskeletal structure that tightly wraps around the nucleus of a wide range of somatic cells. Fibers in the actin cap are distinct from well-characterized, conventional actin fibers at the basal and dorsal surfaces of adherent cells in their subcellular location, internal organization, dynamics, ability to generate contractile forces, response to cytoskeletal pharmacological treatments, response to biochemical stimuli, regulation by components of the linkers of nucleoskeleton and cytoskeleton (LINC) complexes, and response to disease-associated mutations in LMNA, the gene that encodes for the nuclear lamin component lamin A/C. The perinuclear actin cap precisely shapes the nucleus in interphase cells. The perinuclear actin cap may also be a mediator of microenvironment mechanosensing and mechanotransduction, as well as a regulator of cell motility, polarization and differentiation.

Mapping local matrix remodeling induced by a migrating tumor cell using three-dimensional multiple-particle tracking.

Mesenchymal cell migration through a three-dimensional (3D) matrix typically involves major matrix remodeling. The direction of matrix deformation occurs locally in all three dimensions, which cannot be measured by current techniques. To probe the local, 3D, real-time deformation of a collagen matrix during tumor cell migration, we developed an assay whereby matrix-embedded beads are tracked simultaneously in all three directions with high resolution. To establish a proof of principle, we investigated patterns of collagen I matrix deformation near fibrosarcoma cells in the absence and presence of inhibitors of matrix metalloproteinases and acto-myosin contractility. Our results indicate that migrating cells show patterns of local matrix deformation toward the cell that are symmetric in magnitude with respect to the axis of cell movement. In contrast, patterns of matrix release from the cell are asymmetric: the matrix is typically relaxed first at the back of the cell, allowing forward motion, and then at the cell's leading edge. Matrix deformation in regions of the matrix near the cell's leading edge is elastic and mostly reversible, but induces irreversible matrix rupture events near the trailing edge. Our results also indicate that matrix remodeling spatially correlates with protrusive activity. This correlation is mediated by myosin II and Rac1, and eliminated after inhibition of pericellular proteolysis or ROCK. We have developed an assay based on high-resolution 3D multiple-particle tracking that allows us to probe local matrix remodeling during mesenchymal cell migration through a 3D matrix and simultaneously monitor protrusion dynamics.