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Abstract DNA is the genetic code found inside all living cells and its molecular stability can also be utilized outside the cell. While extracellular DNA (eDNA) has been identified as a structural polymer in bacterial biofilms, whether it persists stably throughout development remains unclear. Here, we report that eDNA is temporarily invested in the biofilm matrix before being reclaimed later in development. Specifically, by imaging eDNA dynamics within undomesticated Bacillus subtilis biofilms, we found eDNA is produced during biofilm establishment before being globally degraded in a spatiotemporally coordinated pulse. We identified YhcR, a secreted Ca2+-dependent nuclease, as responsible for eDNA degradation in pellicle biofilms. YhcR cooperates with two other nucleases, NucA and NucB, to reclaim eDNA for its phosphate content in colony biofilms. Our results identify extracellular nucleases that are crucial for eDNA reclamation during biofilm development and we therefore propose a new role for eDNA as a dynamic metabolic reservoir.

Abstract Short bowel syndrome (SBS) leads to severe morbidity and mortality. Intestinal adaptation is crucial in improving outcomes. To understand the human gene pathways associated with adaptation, we perform single-cell transcriptomic analysis of human small intestinal organoids explanted from mice with experimental SBS. We show that transmembrane ion pathways, specifically the transepithelial zinc transport pathway genes SLC39A4 and SLC39A5, are upregulated in SBS. This discovery is corroborated by an external dataset, bulk RT-qPCR, and Western blots. Oral zinc supplementation is shown to improve survival and weight gain of SBS mice and increase the proliferation of intestinal crypt cells in vitro. Finally, we identify the upregulation of SLC39A5 and associated transcription factor KLF5 in biopsied intestinal tissue specimens from patients with SBS. Thus, we identify zinc supplementation as a potential therapy for SBS and describe a xenotransplantation model that provides a platform for discovery in other intestinal diseases.

Abstract Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society’s most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales—molecular, circuit/network, cell/cell-free systems, biological communities, and societal—giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists.

Linking administrative records across programs can yield person-centered information, including client characteristics, public service trajectories, and outcomes and help to answer policy-related questions. Several solutions are available for undertaking record linkage, producing linkage keys for merging data sources for positively matched pairs of records. In this session, we will demonstrate a new application of the Python RecordLinkage package to family-based record linkages with machine learning algorithms for probability scoring, which we call probabilistic record linkage for families (PRLF). First, we will demonstrate the utility of PRLF with a simulation of administrative records and assess linkage accuracy with variations in match rates and data degradation. Second, we will compare generalized linear model estimates across three record linkage solutions (PRLF, ChoiceMaker, and Link Plus). Findings from the simulation study indicate linkage accuracy is largely influenced by degradation (e.g., missing data fields, erroneous or incomplete values) compared to the proportion of simulated matches between datasets. Results from the methods comparison using real world data indicate that all three solutions, when optimized, provide similar results for researchers. We discuss the strengths of our process, such as the use of ensemble methods, to improve match accuracy. We then will identify caveats of record linkage in the context of administrative data. The tool was developed in Python to allow for researchers to work with open-source software and adjust the basic workflow to fit their linkage needs. We will identify several partnerships where this collaboration has worked successfully and empower attendees with access to this useful tool.

The [Index] is a simple, yet innovative, tool that makes better use of data routinely collected at birth to summarize the assets that exist across and within communities. Comprising 12 health, family, and financial indicators available on standard birth records, the Index offers a strengths-based, cost-effective, holistic, valid, and meaningful characterization of the conditions into which newborns are born at both a state and local level. Given the strong, graded relationship between [Index] scores and the predicted probability of child protection involvement and death before age 5, the Index has become an important tool among policymakers to identify communities who should be prioritized for investment so that supports can be allocated more equitably and earlier in children’s lives. It also has provided an opportunity to develop a representative sample of children born into families with historically high service needs, but low engagement, to whom we would reach out directly to gather their perspectives of service experiences. This information will be augmented with administrative records as children age. This work will fill a major gap in information about the needs and experiences of the entire population of young children and families – not just those who engage in services – and the barriers, benefits, and challenges new parents may face in accessing the supports they need. This session will highlight the [Index], its relationship to child-level outcomes, how communities are using it to facilitate equitable investment, and, given international birth record standardization, how other jurisdictions might implement a similar tool.

Background & Aims: The abdominal discomfort experienced by patients with colitis may be attributable in part to the presence of small intestinal dysmotility, yet mechanisms linking colonic inflammation with small-bowel motility remain largely unexplored. We hypothesize that colitis results in small intestinal hypomotility owing to a loss of enteroendocrine cells (EECs) within the small intestine that can be rescued using serotonergic-modulating agents. Methods: Male C57BL/6J mice, as well as mice that overexpress (EECOVER) or lack (EECDEL) NeuroD1+ enteroendocrine cells, were exposed to dextran sulfate sodium (DSS) colitis (2.5% or 5% for 7 days) and small intestinal motility was assessed by 70-kilodalton fluorescein isothiocyanate–dextran fluorescence transit. EEC number and differentiation were evaluated by immunohistochemistry, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining, and quantitative reverse-transcriptase polymerase chain reaction. Mice were treated with the 5-hydroxytryptamine receptor 4 agonist prucalopride (5 mg/kg orally, daily) to restore serotonin signaling. Results: DSS-induced colitis was associated with a significant small-bowel hypomotility that developed in the absence of significant inflammation in the small intestine and was associated with a significant reduction in EEC density. EEC loss occurred in conjunction with alterations in the expression of key serotonin synthesis and transporter genes, including Tph1, Ddc, and Slc6a4. Importantly, mice overexpressing EECs revealed improved small intestinal motility, whereas mice lacking EECs had worse intestinal motility when exposed to DSS. Finally, treatment of DSS-exposed mice with the 5-hydroxytryptamine receptor 4 agonist prucalopride restored small intestinal motility and attenuated colitis. Conclusions: Experimental DSS colitis induces significant small-bowel dysmotility in mice owing to enteroendocrine loss that can be reversed by genetic modulation of EEC or administering serotonin analogs, suggesting novel therapeutic approaches for patients with symptomatic colitis.

Summary: Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling.

ABSTRACT Biofilms provide individual bacteria with many advantages, yet dense cellular proliferation can also create intrinsic metabolic challenges including excessive acidification. Because such pH stress can be masked in buffered laboratory media—such as MSgg commonly used to study Bacillus subtilis biofilms—it is not always clear how such biofilms cope with minimally buffered natural environments. Here, we report how B. subtilis biofilms overcome this intrinsic metabolic challenge through an active pH regulation mechanism. Specifically, we find that these biofilms can modulate their extracellular pH to the preferred neutrophile range, even when starting from acidic and alkaline initial conditions, while planktonic cells cannot. We associate this behavior with dynamic interplay between acetate and acetoin biosynthesis and show that this mechanism is required to buffer against biofilm acidification. Furthermore, we find that buffering-deficient biofilms exhibit dysregulated biofilm development when grown in minimally buffered conditions. Our findings reveal an active pH regulation mechanism in B. subtilis biofilms that could lead to new targets to control unwanted biofilm growth.IMPORTANCEpH is known to influence microbial growth and community dynamics in multiple bacterial species and environmental contexts. Furthermore, in many bacterial species, rapid cellular proliferation demands the use of overflow metabolism, which can often result in excessive acidification. However, in the case of bacterial communities known as biofilms, these acidification challenges can be masked when buffered laboratory media are employed to stabilize the pH environment for optimal growth. Our study reveals that B. subtilis biofilms use an active pH regulation mechanism to mitigate both growth-associated acidification and external pH challenges. This discovery provides new opportunities for understanding microbial communities and could lead to new methods for controlling biofilm growth outside of buffered laboratory conditions.

Biomass is an excellent sustainable carbon neutral energy source, however its use as a coal/petroleum coke substitute in thermal applications poses several challenges. Several inherent properties of biomass including higher heating value (HHV), bulk density, and its hydrophilic and fibrous nature, all contribute to challenges for it to be used as a solid fuel. Torrefaction or mild pyrolysis is a well-accepted thermal pretreatment technology that solves most of the above-mentioned challenges and results in a product with superior coal-like properties. Torrefaction involves the heating of biomass to moderate temperatures typically between 200 °C and 300 °C in a non-oxidizing atmosphere. This study focused on evaluating the influence of torrefaction operating temperature (204–304 °C) and residence time (10–40 min) on properties of pine. Tests were performed on a continuous 0.3 ton/day indirectly heated rotary reactor. The influence of torrefaction operational conditions on pine was evaluated in terms of the composition of torrefied solids, mass yield, energy yield, and HHV using a simulated model developed in Aspen Plus™ software. A kinetic model was established based on the experimental data generated. An increase in torrefaction severity (increasing temperature and residence time) resulted in an increase in carbon content, accompanied with a decrease in oxygen and hydrogen. Results from the simulated model suggest that the solid and energy yields decreased with an increase in temperature and residence time. Solid yield varied from 80% at 204 °C to 68% at 304 °C, and energy yield varied from 99% at 204 °C to 70% at 304 °C, respectively. On the other hand, HHV improved from 22.8 to 25.1 MJ/kg with an increase in temperature at 20 min residence time. Over the range of 10 to 40 min residence time at 260 °C, solid and energy yields varied from 77% to 59% and 79% to 63%, respectively; however the HHV increased by only 3%. Solid yield, energy yield, and HHV simulated data were within the 5% error margin when compared to the experimental data. Validation of the simulation parameters was achieved by the conformance of the experimental and simulation data obtained under the same testing conditions. These simulated parameters can be utilized to study other operating conditions fundamental for the commercialization of these processes. Desirable torrefaction temperature to achieve the highest solid fuel yield can be determined using the energy yield and mass loss data.

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.

Child maltreatment is a pervasive public health problem with well-documented impacts on a child's well-being. The connection between social determinants of health and child maltreatment is well established, particularly economic [...]

Background In 2011, California enacted its public safety realignment initiative (realignment) motivated by a U.S. Supreme Court ruling to reduce state prison overcrowding and in effort to reduce recidivism. Realignment transferred authority for lower-level felony offenders from the state to the counties, leading to a rapid reduction in state prison incarceration levels. Objective This study drew on a unique dataset to assess the effects of California's efforts to downsize the prison system on maternal incarceration levels and to better understand the characteristics of incarcerated mothers and their children. Methods Incarceration records concerning all women in California state prisons between 2010 and 2012 (N = 16,917) were linked to 7.5 million vital birth records dating to 1999 to identify incarcerated women who had given birth. Multinomial logistic regression models were specified to better understand offense type differences among incarcerated mothers versus nonmothers. Results Findings indicate that realignment disproportionately affected women. The number of men entering prison decreased 67.8% between 2010 and 2012. In comparison, the number of women entering prison decreased 78.5%. Further, more than half of incarcerated women had given birth. Mothers were more likely than nonmothers to be convicted of nonviolent crimes. Discussion This study underscores how prison downsizing can disproportionately reduce incarceration levels for women. Given that such large proportion of incarcerated women were mothers, this policy change may have potential spillover next-generation benefits. Finally, this work reinforces the potential to use linked administrative records to study incarcerated populations., Author(s): John Prindle [sup.1] , Andrea Lane Eastman [sup.1] , Qianwei Zhao [sup.2] , Mia Bird [sup.3] , Emily Putnam-Hornstein [sup.1] [sup.4] Author Affiliations: (1) grid.42505.36, 0000 0001 2156 6853, [...]

Research with administrative records involves the challenge of limited information in any single data source to answer policy-related questions. Record linkage provides researchers with a tool to supplement administrative datasets with other information about the same people when identified in separate sources as matched pairs. Several solutions are available for undertaking record linkage, producing linkage keys for merging data sources for positively matched pairs of records. In the current manuscript, we demonstrate a new application of the Python RecordLinkage package to family-based record linkages with machine learning algorithms for probability scoring, which we call probabilistic record linkage for families (PRLF). First, a simulation of administrative records identifies PRLF accuracy with variations in match and data degradation percentages. Accuracy is largely influenced by degradation (e.g., missing data fields, mismatched values) compared to the percentage of simulated matches. Second, an application of data linkage is presented to compare regression model estimate performance across three record linkage solutions (PRLF, ChoiceMaker, and Link Plus). Our findings indicate that all three solutions, when optimized, provide similar results for researchers. Strengths of our process, such as the use of ensemble methods, to improve match accuracy are discussed. We then identify caveats of record linkage in the context of administrative data.

Signal transmission among cells enables long-range coordination in biological systems. However, the scarcity of quantitative measurements hinders the development of theories that relate signal propagation to cellular heterogeneity and spatial organization. We address this problem in a bacterial community that employs electrochemical cell-to-cell communication. We developed a model based on percolation theory, which describes how signals propagate through a heterogeneous medium. Our model predicts that signal transmission becomes possible when the community is organized near a critical phase transition between a disconnected and a fully connected conduit of signaling cells. By measuring population-level signal transmission with single-cell resolution in wild-type and genetically modified communities, we confirm that the spatial distribution of signaling cells is organized at the predicted phase transition. Our findings suggest that at this critical point, the population-level benefit of signal transmission outweighs the single-cell level cost. The bacterial community thus appears to be organized according to a theoretically predicted spatial heterogeneity that promotes efficient signal transmission.

ABSTRACT The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by Gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations and freely diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilized three-dimensional (3D) single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the enhanced yellow fluorescent protein (eYFP)-labeled ATPase YeSctN and its regulator, YeSctL, revealed that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed both in wild-type cells and in ΔsctD mutants, which cannot assemble injectisomes. In ΔsctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding, which has important implications for how injectisomes are functionally regulated. IMPORTANCE Injectisomes are membrane-embedded, multiprotein assemblies used by bacterial pathogens to inject virulent effector proteins into eukaryotic host cells. Protein secretion is regulated by cytosolic proteins that dynamically bind and unbind at injectisomes. However, how these regulatory proteins interact with each other remains unknown. By measuring the diffusion rates of single molecules in living cells, we show that cytosolic injectisome proteins form distinct oligomeric complexes with each other prior to binding to injectisomes. We additionally identify the molecular compositions of these complexes and quantify their relative abundances. Quantifying to what extent cytosolic proteins exist as part of larger complexes in living cells has important implications for deciphering the complexity of biomolecular mechanisms. The results and methods reported here are thus relevant for advancing our understanding of how injectisomes and related multiprotein assemblies, such as bacterial flagellar motors, are functionally regulated.

Most biological processes in living cells rely on interactions between proteins. Live-cell compatible approaches that can quantify to what extent a given protein participates in homo- and hetero-oligomeric complexes of different size and subunit composition are therefore critical to advance our understanding of how cellular physiology is governed by these molecular interactions. Biomolecular complex formation changes the diffusion coefficient of constituent proteins, and these changes can be measured using fluorescence microscopy-based approaches, such as single-molecule tracking, fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching. In this review, we focus on the use of single-molecule tracking to identify, resolve, and quantify the presence of freely-diffusing proteins and protein complexes in living cells. We compare and contrast different data analysis methods that are currently employed in the field and discuss experimental designs that can aid the interpretation of the obtained results. Comparisons of diffusion rates for different proteins and protein complexes in intracellular aqueous environments reported in the recent literature reveal a clear and systematic deviation from the Stokes–Einstein diffusion theory. While a complete and quantitative theoretical explanation of why such deviations manifest is missing, the available data suggest the possibility of weighing freely-diffusing proteins and protein complexes in living cells by measuring their diffusion coefficients. Mapping individual diffusive states to protein complexes of defined molecular weight, subunit stoichiometry, and structure promises to provide key new insights into how protein–protein interactions regulate protein conformational, translational, and rotational dynamics, and ultimately protein function. [ABSTRACT FROM AUTHOR]