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.

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.

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.

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.

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.

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.

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.

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.

Nutrient transport, shear strength and hydraulic characteristics of topsoils amended with mulch, compost and biosolids.

Anthropogenic disturbance of soils can disrupt soil structure, diminish fertility, alter soil chemical properties, and cause erosion. Current remediation practices involve amending degraded urban topsoils lacking in organic matter and nutrition with organic amendments (OA) to enhance vegetative growth. However, the impact of OAs on water quality and structural properties at rates that meet common topsoil organic matter specifications need to be studied and understood. This study tested three commonly available OAs: shredded wood mulch, leaf-based compost, and class A Exceptional Quality stabilized sewage sludge (or biosolids) for nutrient (nitrogen and phosphorus) water quality, soil shear strength, and hydraulic properties, through two greenhouse tub studies. Findings showed that nitrogen losses to leachate were greater in the biosolids amended topsoils compared to leaf-compost, mulch amended topsoils, and control treatments. Steady-state mean total nitrogen (N) concentrations from biosolids treatment exceeded typical highway stormwater concentrations by at least 25 times. Soil total N content combined with the carbon:nitrogen ratio were identified to be the governing properties of N leaching in soils. Study soils, irrespective of the type of amendment, reduced the applied (tap) water phosphorus (P) concentration of ∼0.3 mg-P/L throughout the experiment. Contrary to the effects on N leaching, P was successfully retained by the biosolids amendment, due to the presence of greater active iron contents. A breakthrough mechanism for P was observed in leaf compost amended soil, where the effluent concentrations of P continued to increase with each rainfall application, possibly due to an saturation of soil adsorption sites. The addition of OAs also improved the strength and hydraulic properties of soils. The effective interlocking mechanisms between the soil and OA surfaces could provide soil its required strength and stability, particularly on slopes. OAs also improved soil fertility to promote turf growth. Presence of vegetative root zones can further reinforce the soil and control erosion.

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.

Light-based juxtacrine signaling between synthetic cells.

Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with different types of responses. Here, we design and demonstrate a light-activated contact-dependent communication tool for synthetic cells. We utilize a split bioluminescent protein to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. Our modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

Interactions of particulate- and dissolved-phase heavy metals in a mature stormwater bioretention cell.

Bioretention is an increasingly common stormwater control measure (SCM) for mitigation of stormwater quantity and quality. Studies from lab to field scale have shown successful removal of total metals from stormwater, especially Cu and Zn which are ubiquitous in the urban environment yet detrimental to aquatic ecosystems. While bioretention effectively removes particulate matter and particulate bound (PB) contaminants, removal performance of dissolved metals has been neglected in field studies. After approximately two decades of these systems being implemented, with a typical design-life of 20 years, performance of mature systems is unknown. This study examined the performance of a 16- to 18-year-old bioretention cell by characterizing Cu and Zn partitioning and removal. Flow-weighted composite samples of stormwater and bioretention effluent were collected and analyzed for total and dissolved metals. Size-fractioned road-deposited sediments (RDS) were collected and analyzed for metals and particle size distribution. The comparison of RDS and PB metals showed that PB-Zn was enriched in stormwater, indicating higher mobility of PB-Zn compared to PB-Cu. The mature bioretention system effectively removed particulates and PB-metals with average load reductions of 82% and 83%, respectively. While concentrations for dissolved metals were low (<40 µg/L), no significant difference between influent and effluent was observed. Effluent concentrations of total and dissolved Cu, total organic carbon, and particulates were not significantly different from those measured over 10 years ago at the site, while total Zn effluent concentration slightly increased. MINTEQ speciation modeling showed that Cu was approximately 100% bound with dissolved organic matter (DOM) in all bioretention effluent. While Zn was also mostly bound with DOM in effluent, some events showed free ionic Zn reaching concentrations in the same order of magnitude. Media amendments, maintenance, and monitoring of SCMs should be considered where further removal of dissolved metals is necessary for the protection of aquatic environments.

Development of mechanosensitive synthetic cells for biomedical applications.

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from mechanosensitive synthetic cells for disease treatments.

Impact of COVID-19 pandemic on social isolation and loneliness among minority populations.

BACKGROUND: The impact of social isolation and loneliness (SIL) was heightened during the COVID-19 pandemic. Although the pandemic disproportionately affected racial/ ethnic minorities, no studies have investigated the ramifications of the pandemic on SIL among these populations. This study aimed to determine the prevalence and pervasiveness of SIL during the COVID-19 pandemic on minority communities. MATERIALS AND METHODS: This was a single center, cross sectional study conducted by scientists from the University of Rochester Medical Center (URMC) working in collaboration with members of the Rochester community. Adult patients presenting to the emergency department at URMC who identified themselves as belonging to minority communities were asked to complete a survey that comprised questions from the Lubben Social Network Scale-6 and questions from the Campaign to End Loneliness Measurement Tool. We analyzed the percentage of SIL and conducted linear regression models to study the association between these outcomes and race/ ethnicity, age, gender, chronic disease status and the frequency of hospitalizations. RESULTS: A total of 1,029 subjects completed the survey. Social isolation was reported by 375 (37%) persons. Those of Latinx ethnicity had higher prevalence of social isolation (41%) compared to those of Black/African American race (36%) and also had higher degrees of isolation (14.8%) (15.42; p = 0.07). Loneliness was documented by 215 (21%) for the cohort with no differences based on race or ethnicity. CONCLUSIONS: Social isolation was common among minority communities during the pandemic but loneliness was less pervasive. The study highlights the need to address the specific needs of these populations.

Firearm Violence Involving Adults 65 and Older During the Novel Coronavirus Disease Pandemic.

BACKGROUND: Rates of firearm violence (FV) surged during the COVID-19 pandemic. However, there is a paucity of data regarding older adults (OAs) (≥65 years old). This study aimed to evaluate patterns of FV against OAs before and after the COVID-19 pandemic, hypothesizing decreased firearm incidents, injuries, and deaths for OAs due to restricted social movement. METHODS: Retrospective (2016-2021) data for OAs were obtained from the Gun Violence Archive. The rate of FV was weighted per 10,000 OAs using annual population data from the United States Census Bureau. Mann-Whitney U tests were performed to compare annual firearm incidence rates, number of OAs killed, and number of OAs injured from 2016-2020 to 2021. RESULTS: From 944 OA-involved shootings, 842 died in 2021. The median total firearm incidents per month per 10,000 OAs decreased in 2021 vs 2016 (.65 vs .38, P < .001), 2017 (.63 vs .38, P < .001), 2018 (.61 vs .38, P < .001), 2019 (.39 vs .38, P = .003), and 2020 (.43 vs .38, P = .012). However, there was an increased median number of OAs killed in 2021 vs 2020 (.38 vs .38, P = .009), but no difference from 2016-2019 vs 2021 (all P > .05). The median number of firearm injuries decreased from 2017 to 2021 (.21 vs .19, P = .001) and 2020 to 2021 (.19 vs .19 P < .001). DISCUSSION: Firearm incidents involving OAs decreased in 2021 compared to pre-pandemic years; however, there was a slight increase in deaths compared to 2020. This may reflect increased social isolation; however, future research is needed to understand why this occurred.

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.

Behavioral Impairments and Increased Risk of Cortical Atrophy Risk Scores Among World Trade Center Responders.

Objective: World Trade Center (WTC) responders are susceptible to both cognitive and neuropsychiatric impairments, particularly chronic posttraumatic stress disorder. The present study examined self-reported behavioral impairments in a sample of 732 WTC responders, 199 of whom were determined to have high risk of WTC-related cortical atrophy by an artificial neural network. Results: We found that responders at increased risk of cortical atrophy showed behavioral impairment across five domains: motivation, mood, disinhibition, empathy, and psychosis (14.6% vs 3.9% in the low-risk group; P = 3.90 × 10-7). Factor analysis models revealed that responders at high risk of cortical atrophy tended to have deficits generalized across all aspects of behavioral impairment with focal dysfunction in sensory psychosis. We additionally describe how relationships are modulated by exposure severity and pharmacological treatments. Discussion: Our findings suggest a potential link between sensory deficits and the development of cortical atrophy in WTC responders and may indicate symptoms consistent with a clinical portrait of parietal dominant Alzheimer's disease or a related dementia (ADRD). Results underscore the importance of investigating neuropsychiatric symptomatology in clinical evaluations of possible ADRD.