Extracellular electron uptake (EEU) is the ability of microbes to take up electrons from solid-phase conductive substances such as metal oxides. EEU is performed by prevalent phototrophic bacterial ...genera, but the electron transfer pathways and the physiological electron sinks are poorly understood. Here we show that electrons enter the photosynthetic electron transport chain during EEU in the phototrophic bacterium Rhodopseudomonas palustris TIE-1. Cathodic electron flow is also correlated with a highly reducing intracellular redox environment. We show that reducing equivalents are used for carbon dioxide (CO
) fixation, which is the primary electron sink. Deletion of the genes encoding ruBisCO (the CO
-fixing enzyme of the Calvin-Benson-Bassham cycle) leads to a 90% reduction in EEU. This work shows that phototrophs can directly use solid-phase conductive substances for electron transfer, energy transduction, and CO
fixation.
3D printing has the potential to deliver personalized implants and devices for obstetric and gynecologic applications. The aim of this study is to engineer customizable and biodegradable 3D printed ...implant materials that can elute estrogen and/or progesterone. All 3D constructs were printed using polycaprolactone (PCL) biodegradable polymer laden with estrogen or progesterone and were subjected to hormone-release profile studies using ELISA kits. Material thermal properties were tested using thermogravimetric analysis and differential scanning calorimetry. The 3D printed constructs showed extended hormonal release over a one week period. Cytocompatibility and bioactivity were assessed using a luciferase assay. The hormone-laden 3D printed constructs demonstrated an increase in luciferase activity and without any deleterious effects. Thermal properties of the PCL and hormones showed degradation temperatures above that of the temperature used in the additive manufacturing process-suggesting that 3D printing can be achieved below the degradation temperatures of the hormones. Sample constructs in the shape of surgical meshes, subdermal rods, intrauterine devices and pessaries were designed and printed. 3D printing of estrogen and progesterone-eluting constructs was feasible in this proof of concept study. These custom designs have the potential to act as a form of personalized medicine for drug delivery and optimized fit based on patient-specific anatomy.
Photoferrotrophy is a form of anoxygenic photosynthesis whereby bacteria utilize soluble or insoluble forms of ferrous iron as an electron donor to fix carbon dioxide using light energy. They can ...also use poised electrodes as their electron donor via phototrophic extracellular electron uptake (phototrophic EEU). The electron uptake mechanisms underlying these processes are not well understood. Using
TIE-1 as a model, we show that a single periplasmic decaheme cytochrome
, PioA, and an outer membrane porin, PioB, form a complex allowing extracellular electron uptake across the outer membrane from both soluble iron and poised electrodes. We observe that PioA undergoes postsecretory proteolysis of its N terminus to produce a shorter heme-attached PioA (holo-PioA
, where PioA
represents the C terminus of PioA), which can exist both freely in the periplasm and in a complex with PioB. The extended N-terminal peptide controls heme attachment, and its processing is required to produce wild-type levels of holo-PioA
and holo-PioA
B complex. It is also conserved in PioA homologs from other phototrophs. The presence of PioAB in these organisms correlate with their ability to perform photoferrotrophy and phototrophic EEU.
Some anoxygenic phototrophs use soluble iron, insoluble iron minerals (such as rust), or their proxies (poised electrodes) as electron donors for photosynthesis. However, the underlying electron uptake mechanisms are not well established. Here, we show that these phototrophs use a protein complex made of an outer membrane porin and a periplasmic decaheme cytochrome (electron transfer protein) to harvest electrons from both soluble iron and poised electrodes. This complex has two unique characteristics: (i) it lacks an extracellular cytochrome
, and (ii) the periplasmic decaheme cytochrome
undergoes proteolytic cleavage to produce a functional electron transfer protein. These characteristics are conserved in phototrophs harboring homologous proteins.
We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface ...acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular alga
is widely used to investigate the mechanisms underlying ciliary beating and coordination. However, freely swimming cells are difficult to image with sufficient resolution to capture cilia motion, necessitating that the cell body be held during experiments. Acoustic confinement is a compelling alternative to use of a micropipette, or to magnetic, electrical, and optical trapping that may modify the cells and affect their behavior. Beyond establishing our approach to studying microswimmers, we demonstrate a unique ability to mechanically perturb cells via rapid acoustic positioning.
ObjectiveThe COVID-19 pandemic has precipitated widespread shortages of filtering facepiece respirators (FFRs) and the creation and sharing of proposed substitutes (novel designs, repurposed ...materials) with limited testing against regulatory standards. We aimed to categorically test the efficacy and fit of potential N95 respirator substitutes using protocols that can be replicated in university laboratories.SettingAcademic medical centre with occupational health-supervised fit testing along with laboratory studies.ParticipantsSeven adult volunteers who passed quantitative fit testing for small-sized (n=2) and regular-sized (n=5) commercial N95 respirators.MethodsFive open-source potential N95 respirator substitutes were evaluated and compared with commercial National Institute for Occupational Safety and Health (NIOSH)-approved N95 respirators as controls. Fit testing using the 7-minute standardised Occupational Safety and Health Administration fit test was performed. In addition, protocols that can be performed in university laboratories for materials testing (filtration efficiency, air resistance and fluid resistance) were developed to evaluate alternate filtration materials.ResultsAmong five open-source, improvised substitutes evaluated in this study, only one (which included a commercial elastomeric mask and commercial HEPA filter) passed a standard quantitative fit test. The four alternative materials evaluated for filtration efficiency (67%–89%) failed to meet the 95% threshold at a face velocity (7.6 cm/s) equivalent to that of a NIOSH particle filtration test for the control N95 FFR. In addition, for all but one material, the small surface area of two 3D-printed substitutes resulted in air resistance that was above the maximum in the NIOSH standard.ConclusionsTesting protocols such as those described here are essential to evaluate proposed improvised respiratory protection substitutes, and our testing platform could be replicated by teams with similar cross-disciplinary research capacity. Healthcare professionals should be cautious of claims associated with improvised respirators when suggested as FFR substitutes.
The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical ...tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patient-derived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-on-a-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research.
Effective intracellular delivery is a significant impediment to research and therapeutic applications at all processing scales. Physical delivery methods have long demonstrated the ability to deliver ...cargo molecules directly to the cytoplasm or nucleus, and the mechanisms underlying the most common approaches (microinjection, electroporation, and sonoporation) have been extensively investigated. In this review, we discuss established approaches, as well as emerging techniques (magnetofection, optoinjection, and combined modalities). In addition to operating principles and implementation strategies, we address applicability and limitations of various in vitro, ex vivo, and in vivo platforms. Importantly, we perform critical assessments regarding (1) treatment efficacy with diverse cell types and delivered cargo molecules, (2) suitability to different processing scales (from single cell to large populations), (3) suitability for automation/integration with existing workflows, and (4) multiplexing potential and flexibility/adaptability to enable rapid changeover between treatments of varied cell types. Existing techniques typically fall short in one or more of these criteria; however, introduction of micro-/nanotechnology concepts, as well as synergistic coupling of complementary method(s), can improve performance and applicability of a particular approach, overcoming barriers to practical implementation. For this reason, we emphasize these strategies in examining recent advances in development of delivery systems.
Abstract
Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor ‘M1’-type ...to protumor ‘M2’-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolves as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell–cell dynamics at high spatial and temporal resolution. Using our recently developed microphysiological ‘tumor-on-a-chip’ (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells, whereas the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polarized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of C-X-C motif chemokines (CXCL9), CXCL10 and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of matrix metalloproteinase 7 and angiopoietin 2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment.
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