The effect of Ficoll 70 on thermally-induced and chemically-induced conformational transitions of an RTX protein is quantitatively accounted for by a unified excluded volume model.

A unified excluded volume model based upon the effective hard particle approximation is developed and used to quantitatively model previously published experimental measurements of the effect of adding high concentrations of an "inert" polymer, Ficoll 70, on conformational transitions of the toxin protein RCL that are induced by addition of calcium at constant temperature or by increasing temperature in the absence and presence of high calcium concentrations. The best-fit of this model, which accounts quantitatively for all of the published data to within experimental precision, yields an estimate of the volume of solution excluded to Ficoll by each of four identified conformational states of RCL: H - the most compact conformation adopted in the limits of high calcium concentration and low temperature, H* - the conformation adopted in the limits of high calcium concentration and high temperature, A - the conformation adopted in the limits of low (or no) calcium at low temperature, and A* - the conformation adopted in the limits of low calcium and high temperature. Ficoll exclusion volumes increase in the order H < H* < A < A*. These results are discussed in the context of the physiological functions of the RTX proteins, which are involved in the secretion process and the calcium-induced folding of bacterial virulence factors.

Engineering Sequestration-Based Biomolecular Classifiers with Shared Resources.

Constructing molecular classifiers that enable cells to recognize linear and nonlinear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effects of biological constraints such as resource limitation and competitive binding on the function of those classifiers have been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working based on the principles of molecular sequestration between the sigma factor and its antisigma molecule. We then investigate how the output of the biomolecular perceptron, i.e., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multilayer perceptron neural networks and outline design principles that enable the construction of nonlinear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects.

Optimizing native vegetation establishment in urban soils: Assessing the impacts of organic amendments on specific growth parameters.

Following soil disturbances, establishing healthy roadside vegetation can reduce surface water runoff, improve soil quality, decrease erosion, and enhance landscape aesthetics. This study explores the use of organic soil amendments (OAs) as alternatives to conventional vegetation growth approaches, aiming to provide optimal compost mixing ratios for poor soils, and clarify guidelines for OAs' use in roadside projects. Three sandy loam soils and one loam soil were chosen for the study. Organic amendments included yard waste (Y), food waste (F), turkey litter and green waste-based (T) composts, and wood-derived biochar (B). Treatment applications targeted specific increases in the organic matter (OM) percentage of the soils. A selection of seven native species (grasses and forbs) in a total of 156 pots (4 control soils + 4 soils x 4 OAs x 3 application rates, all prepared in triplicates) was used for the pot study experiment. A significant correlation between electrical conductivity (soluble salts) in soil-OA blends and corresponding percent green coverage (%GC) was found. High salts from the T compost either delayed or curtailed growth. Notably, 3 out of the 4 soils amended with biochar exhibited rapid vegetation coverage during initial growth stages compared to other soil-OA blends but reduced the nitrogen (N) uptake and leaf area in black-eyed Susan (BES) plants. In contrast, N uptake was higher in the BES plants emerging from composts T, F, and Y compared to biochar. It is recommended to minimize concentrated manure-based (e.g., turkey litter) composts for roadside projects as an OM source, and alternatively, enriching wood-based biochar with nutrients when used as a soil amendment. Within the current study, composts such as F and Y were well-suited to establish healthy and long-lasting vegetation.

Venoarterial Extracorporeal Membrane Oxygenation Therapy in Patients with Sickle Cell Disease: Case Series and Review for Intensive Care Physicians.

Sickle cell disease (SCD) is associated with substantial morbidity and early mortality in afflicted adults. Cardiopulmonary complications that occur at increased frequency in SCD such as pulmonary embolism, pulmonary arterial hypertension, and acute chest syndrome can acutely worsen right ventricular function and lead to cardiogenic shock. Mechanical circulatory support including venoarterial extracorporeal membrane oxygenation (VA ECMO) is being increasingly utilized to treat hemodynamic collapse in various patient populations. However, a paucity of literature exists to guide the use of mechanical circulatory support in adults with SCD where disease-related sequela and unique hematologic aspects of this disorder may complicate extracorporeal therapy and must be understood. Here, we review the literature and describe three cases of adult patients with SCD who developed cardiogenic shock from acute decompensated right heart failure and were treated clinically with VA ECMO. Using an in vitro ECMO system, we investigate a potential increased risk of systemic fat emboli in patients with SCD who may be experiencing vaso-occlusive events with bone marrow involvement given the high-volume shunting of blood from venous to arterial systems with VA ECMO. The purpose of this study is to describe available extracorporeal life support experiences, review potential complications, and discuss the special considerations needed to further our understanding of the utility of VA ECMO in those with SCD.

Towards a home-grown rural health workforce: Evidence from Tasmania, Australia.

OBJECTIVE: To examine the association between place of origin and principal place of practice (PPP) of domestic Tasmanian health graduates who received end-to-end training with the University of Tasmania (UTAS). METHODS: The 2022 PPP for all UTAS domestic Tasmanian graduates from medicine, nursing, pharmacy, psychology, medical radiation science and paramedicine between 2011 and 2020 was identified using the online Australian Health Practitioner Regulation Agency (Ahpra) registration database. The graduate's place of origin (home address at the time of course application), together with their 2022 PPP, was described using the Modified Monash Model (MM) classification system of remoteness. Data were analysed using STATA. RESULTS: Over the 10-year period, 4079 domestic Tasmanians graduated from health courses at UTAS, of which 3850 (94.4%) were matched to an Australian PPP. In all, 78.3% of graduates were working in Tasmania, while the remainder (21.7%) were employed interstate. Of those with a Tasmanian PPP, 81.4% were working in a regional setting (MM2), while 77.6% of interstate employed graduates recorded a metropolitan (MM1) PPP. Rural place of origin (MM3-7) was associated with rural employment (MM3-7) in both Tasmania (OR, 37.08; 95% CI 29.01-47.39, p < 0.001), and on the Australian mainland (OR, 21.4; 95% CI 17.4-26.3, p < 0.001). CONCLUSIONS: Most domestic Tasmanian origin UTAS health graduates contribute to the state's health workforce after qualifying. Further research is needed to explore PPP over time and to understand why some graduates are motivated to seek employment on the Australian mainland and in particular, metropolitan cities.

Using organic amendments in disturbed soil to enhance soil organic matter, nutrient content and turfgrass establishment.

Disturbed soils, including manufactured topsoils, often lack physical and chemical properties conducive to vegetation establishment. As a result, efforts to stabilize disturbed soils with vegetation are susceptible to failure. Urban organic waste products such as wood mulch, composted leaf and yard waste, and biosolids are widely distributed as organic amendments that enhance sustainability and plant establishment. Correct use can be determined by examining soil properties such as pH; the concentration of soluble salts (SS); and plant available nutrients - particularly N, C and P; as well as root and shoot growth. This research examined the effects of three typical organic amendments on fertility, establishment, and nutrient loss. A manufactured topsoil was used as the base soil for all treatments, including a control unamended soil (CUT), and soil amended with either mulch (MAT), composted leaf and yard waste (LAT), or biosolids (BAT). A 2 % organic matter concentration increase was sought but not achieved due to difficulty in reproducing lab results at a larger scale. Results showed that LAT improved soil fertility, particularly N-P-K concentrations while maintaining a good C:N ratio, pH, and SS concentration. BAT was the most effective at enhancing shoot growth but results suggest that improved growth rates could result in increased maintenance. Additionally, biosolids were an excellent source of nutrients, especially N-P-K and S, but diminished root growth and N leachate losses indicate that N was applied in excess of turfgrass requirements. Therefore, biosolids could be used as fertilizer, subject to recommended rates for turfgrass establishment to prevent poor root growth and waterborne N pollution. To ensure establishment efforts are successful, MAT is not recommended without a supplemental source of soluble N. Altogether, study results and conclusions could inform others seeking to improve specifications for disturbed soil where turfgrass establishment is needed to stabilize soil.

Towards Synthetic Cells with Self-Producing Energy.

Autonomous generation of energy, specifically adenosine triphosphate (ATP), is critical for sustaining the engineered functionalities of synthetic cells constructed from the bottom-up. In this mini-review, we categorize studies on ATP-producing synthetic cells into three different approaches: photosynthetic mechanisms, mitochondrial respiration mimicry, and utilization of non-conventional approaches such as exploiting synthetic metabolic pathways. Within this framework, we evaluate the strengths and limitations of each approach and provide directions for future research endeavors. We also introduce a concept of building ATP-generating synthetic organelle that will enable us to mimic cellular respiration in a simpler way than current strategies. This review aims to highlight the importance of energy self-production in synthetic cells, providing suggestions and ideas that may help overcome some longstanding challenges in this field.

Seeing beyond the blot: A critical look at assumptions and raw data interpretation in Western blotting.

Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in Western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with Western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of Western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

The case for screening for diabetes distress, depression, and anxiety.

Our goal was to determine the prevalence of anxiety and depression in a sample of U.S. military veterans with type 2 diabetes and elevated diabetes distress (DD). Cross-sectional analyses were conducted. The association between DD and anxiety and depression was assessed with logistic regression. Almost 80% of persons with elevated DD had clinically significant anxiety or depression symptoms. The odds of depression and anxiety increased with DD severity. Given the large overlap of depression and anxiety with elevated DD, we recommend providers screen for all three conditions and, if positive, connect to resources for diabetes self-management and/or clinical treatment.

Presentation and Management of Acute Mania in Fanconi-Bickel Syndrome, A Metabolic Genetic Disorder.

Fanconi-Bickel syndrome (FBS) is a rare metabolic disorder caused by decreased glucose transporter 2 (GLUT2) function due to several known mutations in the SLC2A2 gene. As of 2020, 144 cases of FBS have been described in the literature. Metabolic and somatic sequelae include dysglycemia and accumulation of glycogen in the kidney and liver. However, there are no descriptions in the literature of possible neuropsychiatric manifestations of FBS. This case report is to our knowledge the first in this regard, describing a patient with FBS who was admitted to our psychiatric inpatient unit while experiencing acute mania. We conceptualize the case as a novel psychiatric presentation of acute mania in FBS, which may inform our understanding of bipolar disorder pathophysiology because of the hypothesized functional changes in neural pathways involving the paraventricular thalamus induced by decreased GLUT2 activity in FBS.

Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

Cellular mechanotransduction of human osteoblasts in microgravity.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, 4 microfluidic chips capable of measuring single-cell mechanics of hFOBs via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. Our analysis revealed slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell behavior and signaling in space.

A Mammalian-Based Synthetic Biology Toolbox to Engineer Membrane-Membrane Interfaces.

Intercellular membrane-membrane interfaces are compartments with specialized functions and unique biophysical properties that are essential in numerous cellular processes including cell signaling, development, and immunity. Using synthetic biology to engineer or to create novel cellular functions in the intercellular regions has led to an increasing need for a platform that allows generation of functionalized intercellular membrane-membrane interfaces. Here, we present a synthetic biology platform to engineer functional membrane-membrane interfaces using a pair of dimerizing proteins in both cell-free and cellular environments. We envisage this platform to be a helpful tool for synthetic biologists who wish to engineer novel intercellular signaling and communication systems.

Matrix confinement modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites. STATEMENT OF SIGNIFICANCE: The mechanical properties of the tumor microenvironment significantly influence cancer cell migration within the primary tumor, yet how these properties affect intercellular interactions in heterogeneous tumors is not well understood. By utilizing calcium and calcium chelators, we dynamically alter collagen-alginate hydrogel stiffness and investigate tumor cell behavior within co-culture spheroids in response to varying degrees of matrix confinement. High confinement is found to trigger cell sorting while reducing confinement for sorted spheroids facilitates collective cell invasion. Notably, without prior sorting, spheroids do not exhibit burst-like migration, regardless of confinement levels. This work establishes that matrix confinement and intercellular adhesion regulate 3D spheroid dynamics, offering insights into cellular organization and migration within the primary tumor.

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Cellular mechanotransduction of human osteoblasts in microgravity.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, microfluidic chips capable of measuring single-cell mechanics via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. We found slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell signaling in space.

Rearrangement of GUV-confined actin networks in response to micropipette aspiration.

Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures reorganize under applied forces is not entirely understood. Recently, bottom-up reconstitution has enabled studies where dynamic and phenotypic characteristics of various actin networks can be recreated in an isolated cell-like environment. Here, by creating a giant unilamellar vesicle (GUV)-based cell model encapsulating actin networks, we investigate how actin networks rearrange in response to localized stresses applied by micropipette aspiration. We reconstitute actin bundles and branched bundles in GUVs separately and mechanically perturb them. Interestingly, we find that, when aspirated, protrusive actin bundles that are otherwise randomly oriented in the GUV lumen collapse and align along the axis of the micropipette. However, when branched bundles are aspirated, the network remains intact and outside of the pipette while the GUV membrane is aspirated into the micropipette. These results reveal distinct responses in the rearrangement of actin networks in a network architecture-dependent manner when subjected to physical forces.

The bradykinin-forming cascade in anaphylaxis and ACE-inhibitor induced angioedema/airway obstruction.

Anaphylaxis is a potentially life-threatening multi-system allergic reaction to a biological trigger resulting in the release of potent inflammatory mediators from mast cells and basophils and causing symptoms in at least two organ systems that generally include skin, lungs, heart, or gastrointestinal tract in any combination. One exception is profound hypotension as an isolated symptom. There are two types of triggers of anaphylaxis: immunologic and non-Immunologic. Immunologic anaphylaxis is initiated when a foreign antigen directly binds to IgE expressed on mast cells or basophils and induces the release of histamine and other inflammatory substances resulting in vasodilation, vascular leakage, decreased peripheral vascular resistance, and heart muscle depression. If left untreated, death by shock (profound hypotension) or asphyxiation (airway obstruction) can occur. The non-immunologic pathway, on the other hand, can be initiated in many ways. A foreign substance can directly bind to receptors of mast cells and basophils leading to degranulation. There can be immune complex activation of the classical complement cascade with the release of anaphylatoxins C3a and C5a with subsequent recruitment of mast cells and basophils. Finally, hyperosmolar contrast agents can cause blood cell lysis, enzyme release, and complement activation, resulting in anaphylactoid (anaphylactic-like) symptoms. In this report we emphasize the recruitment of the bradykinin-forming cascade in mast cell dependent anaphylactic reactions as a potential mediator of severe hypotension, or airway compromise (asthma, laryngeal edema). We also consider airway obstruction due to inhibition of angiotensin converting enzyme with a diminished rate of endogenous bradykinin metabolism, leading not only to laryngeal edema, but massive tongue swelling with aspiration of secretions.