A versatile active learning workflow for optimization of genetic and metabolic networks.

Optimization of biological networks is often limited by wet lab labor and cost, and the lack of convenient computational tools. Here, we describe METIS, a versatile active machine learning workflow with a simple online interface for the data-driven optimization of biological targets with minimal experiments. We demonstrate our workflow for various applications, including cell-free transcription and translation, genetic circuits, and a 27-variable synthetic CO2-fixation cycle (CETCH cycle), improving these systems between one and two orders of magnitude. For the CETCH cycle, we explore 1025 conditions with only 1,000 experiments to yield the most efficient CO2-fixation cascade described to date. Beyond optimization, our workflow also quantifies the relative importance of individual factors to the performance of a system identifying unknown interactions and bottlenecks. Overall, our workflow opens the way for convenient optimization and prototyping of genetic and metabolic networks with customizable adjustments according to user experience, experimental setup, and laboratory facilities.

Antibody Conjugation of Fluorescent Nanodiamonds for Targeted Innate Immune Cell Activation.

BACKGROUND: fluorescent nanodiamonds (FND) are nontoxic, infinitely photostable nanoparticles that emit near-infrared fluorescence and have a modifiable surface allowing for the generation of protein-FND conjugates. FND-mediated immune cell targeting may serve as a strategy to visualize immune cells and promote immune cell activation. METHODS: uncoated-FND (uFND) were fabricated, coated with glycidol (gFND), and conjugated with immunoglobulin G (IgG-gFND). In vitro studies were performed using a breast cancer/natural killer/monocyte co-culture system, and in vivo studies were performed using a breast cancer mouse model. RESULTS: in vitro studies demonstrated the targeted immune cell uptake of IgG-gFND, resulting in significant immune cell activation and no compromise in immune cell viability. IgG-gFND remained at the tumor site following intratumoral injection compared to uFND which migrated to the liver and kidneys. CONCLUSION: antibody-conjugated FND may serve as immune drug delivery vehicles with "track and trace capabilities" to promote directed antitumor activity and minimize systemic toxicities.

Antimicrobial Drug-Resistant Gram-Negative Saprophytic Bacteria Isolated from Ambient, Near-Shore Sediments of an Urbanized Estuary: Absence of ß-Lactamase Drug-Resistance Genes.

We assessed the prevalence of antimicrobial resistance and screened for clinically relevant ß-lactamase resistance determinants in Gram-negative bacteria from a large urbanized estuary. In contrast to the broad literature documenting potentially hazardous resistance determinants near wastewater treatment discharge points and other local sources of aquatic pollution, we employed a probabilistic survey design to examine ambient, near-shore sediments. We plated environmental samples from 40 intertidal and shallow subtidal areas around San Francisco Bay (California, USA) on drug-supplemented MacConkey agar, and we tested isolates for antimicrobial resistance and presence of clinically relevant ß-lactamase resistance determinants. Of the 74 isolates identified, the most frequently recovered taxa were Vibrio spp. (40%), Shewanella spp. (36%), Pseudomonas spp. (11%), and Aeromonas spp. (4%). Of the 55 isolates tested for antimicrobial resistance, the Vibrio spp. showed the most notable resistance profiles. Most (96%) were resistant to ampicillin, and two isolates showed multidrug-resistant phenotypes: V. alginolyticus (cefotaxime, ampicillin, gentamicin, cefoxitin) and V. fluvialis (cefotaxime, ampicillin, cefoxitin). Targeted testing for class 1 integrons and presence of ß-lactam-resistance gene variants TEM, SHV, OXA, CTX-M, and Klebsiella pneumonia carbapenemase (KPC) did not reveal any isolates harboring these resistance determinants. Thus, while drug-resistant, Gram-negative bacteria were recovered from ambient sediments, neither clinically relevant strains nor mobile ß-lactam resistance determinants were found. This suggests that Gram-negative bacteria in this well-managed, urbanized estuary are unlikely to constitute a major human exposure hazard at this time.

Strongyloidiasis hyperinfection after renal transplant presenting as diffuse alveolar hemorrhage with respiratory failure.

Strongyloides stercoralis is a helminthic enteric parasite estimated to infect at least 30 to 100 million people globally. It is transmitted via contaminated soil with a unique ability to complete its entire life cycle in the human host. It is common in humid, tropical, and subtropical regions of the world and is endemic in the Southeastern United States. Strongyloidiasis hyperinfection has been described in a variety of conditions that impair host immunity, including immunosuppression after transplantation. The syndrome has a high mortality rate but may initially present with nonspecific symptoms. A high degree of clinical suspicion coupled with early detection and aggressive therapeutic measures is paramount to a successful outcome.

Genotypic analysis of uropathogenic Escherichia coli to understand factors that impact the prevalence of ß-lactam-resistant urinary tract infections in a community.

OBJECTIVES: To determine the contribution of specific uropathogenic Escherichia coli (UPEC) lineages, drug resistance genes, and plasmid incompatibility/replicon (Inc) groups to the prevalence of ß-lactam-resistant urinary tract infections (UTIs) in a university community. METHODS: Urine samples were consecutively collected and cultured over a 2-year period from patients presenting to a university health centre with symptoms of UTI. Isolated UPEC were subtyped by multilocus sequence typing and fimH typing, and tested by PCR and sequencing for ß-lactamase genes and plasmid Inc groups. RESULTS: Among 273 UPEC isolates, 85 (31%) were ampicillin-resistant (AMP-R) and 188 (69%) were susceptible to all ß-lactam drugs (AMP-S). Six lineages accounted for two-thirds of the isolates: ST95 (21%), ST69 (11%), ST420 (11%), ST73 (10%), ST127 (8%), and ST404 (3%). ST69 and ST404 were associated with AMP-R (P=0.003, P=0.0005), while ST420 and ST127 were associated with AMP-S (P<0.0001, P=0.027). ST95 contained four fimH types; the ST95/f-6 sublineage was more frequently identified among the AMP-R population (P = 0.009), while the ST95/f-47 sublineage was more frequently identified among the AMP-S population (P=0.007). The most common ß-lactamase gene was blaTEM, which was identified in 81 (95%) AMP-R isolates. IncFIB, IncFIA, and IncB/O type plasmids were the most commonly identified types, and were associated with ß-lactam resistance (P<0.001 for all). CONCLUSIONS: These observations indicate that the prevalence of ß-lactam-resistant UTIs in this community was largely determined by a limited set of circulating UPEC STs and sublineages, carrying TEM ß-lactamase genes that were likely encoded on one of three Inc type plasmids.