Influence of Nonspecific Interactions on Protein Associations: Implications for Biochemistry In Vivo.

The cellular interior is composed of a variety of microenvironments defined by distinct local compositions and composition-dependent intermolecular interactions. We review the various types of nonspecific interactions between proteins and between proteins and other macromolecules and supramolecular structures that influence the state of association and functional properties of a given protein existing within a particular microenvironment at a particular point in time. The present state of knowledge is summarized, and suggestions for fruitful directions of research are offered.

Surfaces as frameworks for intracellular organization.

A large fraction of soluble protein within the interior of living cells may reversibly associate with structural elements, including proteinaceous fibers and phospholipid membranes. In this opinion, we present theoretical and experimental evidence that many of these associations are due to nonspecific attraction between the protein and the surface of the fiber or membrane, and that such associations may lead to substantial changes in the association state of the adsorbed proteins, the biological function of the adsorbed proteins, and the distribution of these proteins between the many microenvironments existing within the cell.

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

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.

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.

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.

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.

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.

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.

Differential regulation of GUV mechanics via actin network architectures.

Actin networks polymerize and depolymerize to construct highly organized structures, thereby endowing the mechanical phenotypes found in a cell. It is generally believed that the amount of filamentous actin and actin network architecture determine cytoplasmic viscoelasticity of the whole cell. However, the intrinsic complexity of a cell and the presence of endogenous cellular components make it difficult to study the differential roles of distinct actin networks in regulating cell mechanics. Here, we model a cell by using giant unilamellar vesicles (GUVs) encapsulating actin filaments and networks assembled by various actin cross-linker proteins. Perturbation of these cytoskeletal vesicles using alternating current electric fields revealed that deformability depends on actin network architecture. While actin-free vesicles exhibited large electromechanical deformations, deformations of GUVs encapsulating actin filaments were significantly dampened. The suppression of electrodeformation of actin-GUVs can be similarly recapitulated by using aqueous poly(ethylene glycol) 8000 solutions at different concentrations to modulate solution viscoelasticity. Furthermore, networks cross-linked by alpha actinin resulted in decreased GUV deformability compared with actin-filament-encapsulating GUVs, and membrane-associated actin networks, through the formation of the dendritic actin cortex, greatly dampened electrodeformation of GUVs. These results highlight that the organization of actin networks regulates the mechanics of GUVs and shed insights into the origin of differential deformability of cells.

Engineering Functional Membrane-Membrane Interfaces by InterSpy.

Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane-membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.

The living interface between synthetic biology and biomaterial design.

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.

Ultrasound simulation training to meet the 2021 Royal College of Radiologists' curriculum for radiology trainees: South East London experience.

AIM: To enhance ultrasound teaching delivery to radiology trainees using a simulation course matched to the 2021 Royal College of Radiologists (RCR) curriculum. MATERIAL AND METHODS: An ultrasound simulation training course was designed for specialty trainees (ST) 1 in radiology, which was based on the 2021 RCR curriculum and covered the top ultrasound training priorities. The course was piloted initially on two occasions in a 1-day format to the August 2021 and the March 2022 ST1 intake trainees. Based on the feedback, a comprehensive 4-day course was developed and delivered between October and December 2022 for the August 2022 ST1 intake, funded by Health Education England. The outcomes measured were subjective trainee feedback using numerical scores and free text. RESULTS: All King's College Hospital NHS Foundation Trust radiology ST1 trainees from the August 2021 to the August 2022 intake participated in ultrasound simulation training. The training matched the RCR curriculum and increased the trainees' confidence and competency in medical ultrasound. CONCLUSIONS: Ultrasound simulation training can be successfully delivered to ST1 trainees to match the 2021 RCR curriculum and enhance training in medical ultrasound for radiologists.

Lysine Ubiquitylation Drives Rhodopsin Protein Turnover.

Rhodopsin is a G-protein-coupled receptor that is specifically and abundantly expressed in rod photoreceptors. Over 150 rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP). The most common mutation in the United States is the conversion of proline to histidine at position 23 (P23H) in the N-terminal domain of rhodopsin. We previously found that P23H rhodopsin was misfolded, ubiquitinylated, and rapidly degraded. Here, we investigated the role of lysine residues on P23H rhodopsin ubiquitinylation and turnover. We transfected HEK293 cells with a P23H human rhodopsin construct where all 11 lysine residues were mutated to arginine (K-null P23H). We found that the K-null P23H rhodopsin was significantly less ubiquitylated than intact P23H rhodopsin. We found that K-null P23H protein turnover was significantly slower compared to P23H rhodopsin through cycloheximide chase analysis. Finally, we also generated a wild-type rhodopsin construct where all lysines were converted to arginine and found significantly reduced ubiquitylation. Our findings identify ubiquitinylation of lysine residues as an important posttranslational modification involved in P23H rhodopsin protein degradation.

Hybrid Vesicles Enable Mechano-Responsive Hydrogel Degradation.

Stimuli-responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano-responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano-responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium-crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel method for engineering mechano-responsive hydrogels that may be useful in various biomedical applications.

In Vitro Synthesis and Reconstitution Using Mammalian Cell-Free Lysates Enables the Systematic Study of the Regulation of LINC Complex Assembly.

Understanding the structure and structure-function relationships of membrane proteins is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional inner nuclear membrane Sad1/UNC-84 (SUN) proteins (i.e., SUN1 and SUN2) in supported lipid bilayers. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and heterotypic interactions between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

Protease activity in single-chain prekallikrein.

Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

Biological nitrogen removal from stormwater in bioretention cells: a critical review.

Excess nitrogen in stormwater degrades surface water quality via eutrophication and related processes. Bioretention has been recognized as a highly effective low-impact development (LID) technology for the management of high runoff volumes and reduction of nitrogen (N) pollutants through various mechanisms. This paper provides a comprehensive and critical review of recent developments on the biological N removal processes occurring in bioretention systems. The key plant- and microbe-mediated N transformation processes include assimilation (N uptake by plants and microbes), nitrification, denitrification, and anammox (anaerobic ammonia oxidation), but denitrification is the major pathway of permanent N removal. Overall, both laboratory- and field-scale bioretention systems have demonstrated promising N removal performance (TN: >70%). The phyla Bacteroidetes and Proteobacteria are the most abundant microbial communities found to be enriched in biofilter media. Furthermore, the denitrifying communities contain several functional genes (e.g., nirK/nirS, and nosZ), and their concentrations increase near the surface of media depth. The N removal effectiveness of bioretention systems is largely impacted by the hydraulics and environmental factors. When a bioretention system operates at: low hydraulic/N loading rate, containing a saturation zone, vegetated with native plants, having deeper and multilayer biofilter media with warm climate temperature and wet storm events periods, the N removal efficiency can be high. This review highlights shortcomings and current knowledge gaps in the area of total nitrogen removal using bioretention systems, as well as identifies future research directions on this topic.

Diagnostic and Imaging Approaches to Chest Wall Lesions.

Chest wall lesions are relatively uncommon and may be challenging once they are encountered on images. Radiologists may detect these lesions incidentally at examinations performed for other indications, or they may be asked specifically to evaluate a suspicious lesion. While many chest wall lesions have characteristic imaging findings that can result in an accurate diagnosis with use of imaging alone, other entities are difficult to distinguish at imaging because there is significant overlap among them. The interpreting radiologist should be familiar with the imaging features of both "do not touch" benign entities (which can be confidently diagnosed with imaging only, with no need for biopsy or resection unless the patient is symptomatic) and lesions that cannot be confidently characterized and thus require further workup. CT and MRI are the main imaging modalities used to assess the chest wall, with each having different benefits and drawbacks. Chest wall lesions can be classified according to their predominant composition: fat, calcification and ossification, soft tissue, or fluid. The identification or predominance of signal intensities or attenuation for these findings, along with the patient age, clinical history, and lesion location, can help establish the appropriate differential diagnosis. In addition, imaging findings in other organs, such as the lungs or upper abdomen, can at times provide clues to the underlying diagnosis. The authors review different chest wall lesions classified on the basis of their composition and highlight the imaging findings that can assist the radiologist in narrowing the differential diagnosis and guiding management. ©RSNA, 2022.

Rapid emergence of life shown by discovery of 3,700-million-year-old microbial structures.

Biological activity is a major factor in Earth's chemical cycles, including facilitating CO2 sequestration and providing climate feedbacks. Thus a key question in Earth's evolution is when did life arise and impact hydrosphere-atmosphere-lithosphere chemical cycles? Until now, evidence for the oldest life on Earth focused on debated stable isotopic signatures of 3,800-3,700 million year (Myr)-old metamorphosed sedimentary rocks and minerals from the Isua supracrustal belt (ISB), southwest Greenland. Here we report evidence for ancient life from a newly exposed outcrop of 3,700-Myr-old metacarbonate rocks in the ISB that contain 1-4-cm-high stromatolites-macroscopically layered structures produced by microbial communities. The ISB stromatolites grew in a shallow marine environment, as indicated by seawater-like rare-earth element plus yttrium trace element signatures of the metacarbonates, and by interlayered detrital sedimentary rocks with cross-lamination and storm-wave generated breccias. The ISB stromatolites predate by 220 Myr the previous most convincing and generally accepted multidisciplinary evidence for oldest life remains in the 3,480-Myr-old Dresser Formation of the Pilbara Craton, Australia. The presence of the ISB stromatolites demonstrates the establishment of shallow marine carbonate production with biotic CO2 sequestration by 3,700 million years ago (Ma), near the start of Earth's sedimentary record. A sophistication of life by 3,700 Ma is in accord with genetic molecular clock studies placing life's origin in the Hadean eon (>4,000 Ma).