A pipeline for malignancy and therapy agnostic assessment of cancer drug response using cell mass measurements.

Functional precision medicine offers a promising complement to genomics-based cancer therapy guidance by testing drug efficacy directly on a patient's tumor cells. Here, we describe a workflow that utilizes single-cell mass measurements with inline brightfield imaging and machine-learning based image classification to broaden the clinical utility of such functional testing for cancer. Using these image-curated mass measurements, we characterize mass response signals for 60 different drugs with various mechanisms of action across twelve different cell types, demonstrating an improved ability to detect response for several slow acting drugs as compared with standard cell viability assays. Furthermore, we use this workflow to assess drug responses for various primary tumor specimen formats including blood, bone marrow, fine needle aspirates (FNA), and malignant fluids, all with reports generated within two days and with results consistent with patient clinical responses. The combination of high-resolution measurement, broad drug and malignancy applicability, and rapid return of results offered by this workflow suggests that it is well-suited to performing clinically relevant functional assessment of cancer drug response.

Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection.

Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB, and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking 10 other AMP genes revealed a role for Cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defense and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks 14 immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

The Drosophila Baramicin polypeptide gene protects against fungal infection.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.

Optical tweezer measurements of asymptotic nonlinearities in complex fluids.

This article presents micro-medium-amplitude oscillatory shear (µMAOS), a method to measure the frequency-dependent micromechanical properties of soft materials in the asymptotically nonlinear regime using optical tweezers. We have developed a theoretical framework to extract these nonlinear mechanical properties of the material from experimental measurements and also proposed a physical interpretation of the third-order nonlinearities measured in single-tone oscillatory tests. We validate the method using a well-characterized surfactant solution of wormlike micelles, and subsequently employ this technique to demonstrate that the cytoplasm of a living cell undergoes strain softening and shear thinning when locally subjected to weakly nonlinear oscillatory deformations.

Prokaryote playhouse: A low-cost, laser-cut acrylic incubator for optogenetic bacterial culture.

Bacterial photography is a printing technique that replaces conventional photochemistry with a living film of engineered Escherichia coli. Many biology teaching labs have adopted monochrome bacterial photography because it offers a captivating playground for illustrating central concepts and lab techniques in biological engineering, particularly in the fields of synthetic biology and optogenetics. Recent improvements have increased the number of color channels from one to three. A key practical challenge in three-color printing is to expose a Petri dish loaded with engineered bacteria to a trichromatic image while maintaining it at 37 °C. Prokaryote Playhouse is a compact, inexpensive, open-source, benchtop incubator for light-sensitive bacterial cultures that makes bacterial photography and similar bacterial optogenetic methods more accessible to teaching labs, makerspaces, and research labs. The system includes a laser-cut, light-tight enclosure; digital thermostat; heated sample shelf; single-board computer; and miniature projector. We built a fleet of Prokaryote Playhouses that students have used to produce hundreds of bacterial photographs in a wide range of educational experiences, ranging from a four-hour introduction to synthetic biology and wet lab techniques to a six-week exploratory class for first-year students at MIT.

The SENIEUR protocol and the efficacy of hepatitis B vaccination in healthy elderly persons by age, gender, and vaccine route.

BACKGROUND: Reduced response to hepatitis B vaccines is associated with aging, confounding and comorbid conditions, as well as inadvertent subcutaneous (SC) inoculation. We hypothesized that the antibody and T cell-mediated immune responses (T-CMI) of elderly adults to a vaccine intended for intramuscular (IM) administration would be attenuated when deposited into SC fat, independent of confounding conditions. RESULTS: Fifty-two healthy, community dwelling elderly adults (65-82 years), seronegative for HBV, were enrolled in the SENIEUR protocol as a strictly healthy population. These seniors were randomized to receive a licensed alum-adjuvanted recombinant HBV vaccine either SC or IM, with the inoculum site verified by imaging. The response rates, defined as hepatitis B surface antibodies (HBsAb) ≥10 IU/L, were significantly lower in the elderly than in young adults, a group of 12, healthy, 21-34-year-old volunteers. Moreover, elderly participants who received the vaccine IM were significantly more likely to be responders than those immunized SC (54% versus 16%, p = 0.008). The low seroconversion rate in the IM group progressively declined with increasing age, and responders had significantly lower HBsAb titers and limited isotype responses. Moreover, T-CMI (proliferation and cytokine production) were significantly reduced in both percentage of responders and intensity of the response for both Th1 and Th2 subsets in the elderly. CONCLUSIONS: Our data demonstrate the blunted immunogenicity of SC inoculation as measured by peak titers and response rates. Further, the qualitative and quantitative deficits in B- and T-CMI responses to primary alum adjuvanted protein antigens persisted even in strictly healthy elderly populations with verified IM placement compared to younger populations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT04162223. Registered 14 November 2019. Retrospectively registered.

The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi.

Fungal infections, widespread throughout the world, affect a broad range of life forms, including agriculturally relevant plants, humans, and insects. In defending against fungal infections, the fruit fly Drosophila melanogaster employs the Toll pathway to induce a large number of immune peptides. Some have been investigated, such as the antimicrobial peptides (AMPs) and Bomanins (Boms); many, however, remain uncharacterized. Here, we examine the role in innate immunity of two related peptides, Daisho1 and Daisho2 (formerly IM4 and IM14, respectively), found in hemolymph following Toll pathway activation. By generating a CRISPR/Cas9 knockout of both genes, Δdaisho, we find that the Daisho peptides are required for defense against a subset of filamentous fungi, including Fusarium oxysporum, but not other Toll-inducible pathogens, such as Enterococcus faecalis and Candida glabrata. Analysis of null alleles and transgenes revealed that the two daisho genes are each required for defense, although their functions partially overlap. Generating and assaying a genomic epitope-tagged Daisho2 construct, we detected interaction in vitro of Daisho2 peptide in hemolymph with the hyphae of F. oxysporum. Together, these results identify the Daisho peptides as a new class of innate immune effectors with humoral activity against a select set of filamentous fungi.

Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

Bombardier Enables Delivery of Short-Form Bomanins in the Drosophila Toll Response.

Toll mediates a robust and effective innate immune response across vertebrates and invertebrates. In Drosophila melanogaster, activation of Toll by systemic infection drives the accumulation of a rich repertoire of immune effectors in hemolymph, including the recently characterized Bomanins, as well as the classical antimicrobial peptides (AMPs). Here we report the functional characterization of a Toll-induced hemolymph protein encoded by the bombardier (CG18067) gene. Using the CRISPR/Cas9 system to generate a precise deletion of the bombardier transcriptional unit, we found that Bombardier is required for Toll-mediated defense against fungi and Gram-positive bacteria. Assaying cell-free hemolymph, we found that the Bomanin-dependent candidacidal activity is also dependent on Bombardier, but is independent of the antifungal AMPs Drosomycin and Metchnikowin. Using mass spectrometry, we demonstrated that deletion of bombardier results in the specific absence of short-form Bomanins from hemolymph. In addition, flies lacking Bombardier exhibited a defect in pathogen tolerance that we trace to an aberrant condition triggered by Toll activation. These results lead us to a model in which the presence of Bombardier in wild-type flies enables the proper folding, secretion, or intermolecular associations of short-form Bomanins, and the absence of Bombardier disrupts one or more of these steps, resulting in defects in both immune resistance and tolerance.

Short-Form Bomanins Mediate Humoral Immunity in Drosophila.

The Bomanins (Boms) are a family of a dozen secreted peptides that mediate the innate immune response governed by the Drosophila Toll receptor. We recently showed that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. Here, we characterize the activity of individual Bom family members. We provide evidence that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. We further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, we find that the level of antifungal activity both in vivo and in vitro is linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) drosomycin and metchnikowin, we find no evidence that Boms act by modifying the expression of the mature forms of these antifungal AMPs.

Allosteric modulation of human dopamine transporter activity under conditions promoting its dimerization.

The human dopamine (DA) transporter (hDAT) is a key regulator of neurotransmission and a target for antidepressants and addictive drugs. Despite the recent resolution of dDAT structures from Drosophila melanogaster, complete understanding of its mechanism of function and even information on its biological assembly is lacking. The resolved dDAT structures are monomeric, but growing evidence suggests that hDAT might function as a multimer, and its oligomerization may be relevant to addictive drug effects. Here, using structure-based computations, we examined the possible mechanisms of hDAT dimerization and its dynamics in a lipid bilayer. Using a combination of site-directed mutagenesis, DA-uptake, and cross-linking experiments that exploited the capacity of Cys-306 to form intermonomeric disulfide bridges in the presence of an oxidizing agent, we tested the effects of mutations at transmembrane segment (TM) 6 and 12 helices in HEK293 cells. The most probable structural model for hDAT dimer suggested by computations and experiments differed from the dimeric structure resolved for the bacterial homolog, LeuT, presumably because of a kink at TM12 preventing favorable monomer packing. Instead, TM2, TM6, and TM11 line the dimer interface. Molecular dynamics simulations of the dimeric hDAT indicated that the two subunits tend to undergo cooperative structural changes, both on local (extracellular gate opening/closure) and global (transition between outward-facing and inward-facing states) scales. These observations suggest that hDAT transport properties may be allosterically modulated under conditions promoting dimerization. Our study provides critical insights into approaches for examining the oligomerization of neurotransmitter transporters and sheds light on their drug modulation.

Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck.

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.

High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays.

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes.

A light-reflecting balloon catheter for atraumatic tissue defect repair.

A congenital or iatrogenic tissue defect often requires closure by open surgery or metallic components that can erode tissue. Biodegradable, hydrophobic light-activated adhesives represent an attractive alternative to sutures, but lack a specifically designed minimally invasive delivery tool, which limits their clinical translation. We developed a multifunctional, catheter-based technology with no implantable rigid components that functions by unfolding an adhesive-loaded elastic patch and deploying a double-balloon design to stabilize and apply pressure to the patch against the tissue defect site. The device uses a fiber-optic system and reflective metallic coating to uniformly disperse ultraviolet light for adhesive activation. Using this device, we demonstrate closure on the distal side of a defect in porcine abdominal wall, stomach, and heart tissue ex vivo. The catheter was further evaluated as a potential tool for tissue closure in vivo in rat heart and abdomen and as a perventricular tool for closure of a challenging cardiac septal defect in a large animal (porcine) model. Patches attached to the heart and abdominal wall with the device showed similar inflammatory response as sutures, with 100% small animal survival, indicating safety. In the large animal model, a ventricular septal defect in a beating heart was reduced to <1.6 mm. This new therapeutic platform has utility in a range of clinical scenarios that warrant minimally invasive and atraumatic repair of hard-to-reach defects.

Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits.

The internal state of an organism influences its perception of attractive or aversive stimuli and thus promotes adaptive behaviors that increase its likelihood of survival. The mechanisms underlying these perceptual shifts are critical to our understanding of how neural circuits support animal cognition and behavior. Starved flies exhibit enhanced sensitivity to attractive odors and reduced sensitivity to aversive odors. Here, we show that a functional remodeling of the olfactory map is mediated by two parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion at the level of the first synapse. Short neuropeptide F sensitizes an antennal lobe glomerulus wired for attraction, while tachykinin (DTK) suppresses activity of a glomerulus wired for aversion. Thus we show parallel neuromodulatory systems functionally reconfigure early olfactory processing to optimize detection of nutrients at the risk of ignoring potentially toxic food resources.

Alternative NF-κB Isoforms in the Drosophila Neuromuscular Junction and Brain.

The Drosophila NF-κB protein Dorsal is expressed at the larval neuromuscular junction, where its expression appears unrelated to known Dorsal functions in embryonic patterning and innate immunity. Using confocal microscopy with domain-specific antisera, we demonstrate that larval muscle expresses only the B isoform of Dorsal, which arises by intron retention. We find that Dorsal B interacts with and stabilizes Cactus at the neuromuscular junction, but exhibits Cactus independent localization and an absence of detectable nuclear translocation. We further find that the Dorsal-related immune factor Dif encodes a B isoform, reflecting a conservation of B domains across a range of insect NF-κB proteins. Carrying out mutagenesis of the Dif locus via a site-specific recombineering approach, we demonstrate that Dif B is the major, if not sole, Dif isoform in the mushroom bodies of the larval brain. The Dorsal and Dif B isoforms thus share a specific association with nervous system tissues as well as an alternative protein structure.

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

High-speed multiple-mode mass-sensing resolves dynamic nanoscale mass distributions.

Simultaneously measuring multiple eigenmode frequencies of nanomechanical resonators can determine the position and mass of surface-adsorbed proteins, and could ultimately reveal the mass tomography of nanoscale analytes. However, existing measurement techniques are slow (<1 Hz bandwidth), limiting throughput and preventing use with resonators generating fast transient signals. Here we develop a general platform for independently and simultaneously oscillating multiple modes of mechanical resonators, enabling frequency measurements that can precisely track fast transient signals within a user-defined bandwidth that exceeds 500 Hz. We use this enhanced bandwidth to resolve signals from multiple nanoparticles flowing simultaneously through a suspended nanochannel resonator and show that four resonant modes are sufficient for determining their individual position and mass with an accuracy near 150 nm and 40 attograms throughout their 150-ms transit. We envision that our method can be readily extended to other systems to increase bandwidth, number of modes, or number of resonators.