Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

Multi-axis fields boost SABRE hyperpolarization.

The inherently low signal-to-noise ratio of NMR and MRI is now being addressed by hyperpolarization methods. For example, iridium-based catalysts that reversibly bind both parahydrogen and ligands in solution can hyperpolarize protons (SABRE) or heteronuclei (X-SABRE) on a wide variety of ligands, using a complex interplay of spin dynamics and chemical exchange processes, with common signal enhancements between 103 and 104. This does not approach obvious theoretical limits, and further enhancement would be valuable in many applications (such as imaging mM concentration species in vivo). Most SABRE/X-SABRE implementations require far lower fields (µT-mT) than standard magnetic resonance (>1T), and this gives an additional degree of freedom: the ability to fully modulate fields in three dimensions. However, this has been underexplored because the standard simplifying theoretical assumptions in magnetic resonance need to be revisited. Here, we take a different approach, an evolutionary strategy algorithm for numerical optimization, multi-axis computer-aided heteronuclear transfer enhancement for SABRE (MACHETE-SABRE). We find nonintuitive but highly efficient multiaxial pulse sequences which experimentally can produce a sevenfold improvement in polarization over continuous excitation. This approach optimizes polarization differently than traditional methods, thus gaining extra efficiency.

Exclusion of latecomers yields a patchwork of viral subpopulations within hosts.

Viruses arriving late to an individual cell are blocked from replicating, an effect called superinfection exclusion. A study in PLOS Biology indicates that this exclusion at the level of individual cells gives rise to a heterogenous landscape of infection within a host.

Publisher Correction: Modular and tunable biological feedback control using a de novo protein switch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Modular and tunable biological feedback control using a de novo protein switch.

De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technology8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.

Binding of the nuclear ribonucleoprotein family member FUS to RNA prevents R-loop RNA:DNA hybrid structures.

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

IMPORTANCE: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

Vibrio cholerae requires oxidative respiration through the bd-I and cbb3 oxidases for intestinal proliferation.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.

Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission.

Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing and high inoculation dose. However, contrary to expectation, trends observed with sub-optimal exposure timing and lower inoculation dose suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

Can the Dose of Belimumab be Reduced in Patients with Systemic Lupus Erythematosus?

OBJECTIVES: The aims of this study were to investigate the prevalence of dose reduction in patients with SLE treated with belimumab (BEL) in Spain, analyze treatment modalities, and determine impact on control of disease activity. METHODS: Retrospective longitudinal and multicentre study of SLE patients treated with BEL. Data on disease activity, treatments and outcomes were recorded before and after reduction (6-12 months), and they were compared. RESULTS: A total of 324 patients were included. The dose was reduced in 29 patients (8.9%). The dosing interval was increased in 9 patients receiving subcutaneous BEL and in 6 patients receiving intravenous BEL. The dose per administration was reduced in 16 patients.Pre-reduction status was remission (2021 DORIS) in 15/26 patients (57.7%) and LLDAS in 23/26 patients (88.5%). After reduction, 2/24 patients (8.3%) and 3/22 patients (13.6%) lost remission at 6 months and 12 months, respectively (not statistically significant [NS]). As for LLDAS, 2/23 patients (8.7%) and 2/21 patients (9.5%) lost their status at 6 and 12 months, respectively (NS). Significantly fewer patients were taking glucocorticoids (GCs) at their 12-month visit, although the median dose of GCs was higher at the 12-month visit (5 [0.62-8.75] vs 2.5 [0-5] at baseline). CONCLUSION: Doses of BEL can be reduced with no relevant changes in disease activity-at least in the short term-in a significant percentage of patients, and most maintain the reduced dose. However, increased clinical or serologic activity may be observed in some patients. Consequently, tighter post-reduction follow-up is advisable.

Preservatives for postmortem brain tissue in biomechanical testing: A pilot study.

Postmortem human subject (PMHS) studies are essential to brain injury research in motor vehicle safety. However, postmortem deterioration reduces the similarity between postmortem test results and in vivo response in material testing of brain tissue and in biomechanical testing of the whole head. This pilot study explores the effect of potential preservatives on brain tissue breakdown to identify promising preservatives that warrant further investigation. To identify preservatives with potential to slow postmortem degradation, samples from an initial PMHS were refrigerated at 10°C to qualitatively compare tissue breakdown from 58 to 152 h postmortem after storage in candidate solutions. On brain tissue samples from a second PMHS, compressive stiffness was measured on six samples immediately after harvest for comparison to the stiffness of 23 samples that were stored at 10°C in candidate solutions for 24 h after harvest. The candidate solutions were artificial cerebrospinal fluid (ACSF) without preservatives; ACSF with a combination of antibiotics and antifungal agents; ACSF with added sodium bicarbonate; and ACSF with both the antibiotic/antifungal combination and sodium bicarbonate. Results were analyzed using multiple linear regression of specimen stiffness on harvest lobe and storage solution to investigate potential differences in tissue stiffness. Qualitative evaluation suggested that samples stored in a solution that contained both the antibiotic/antifungal combination and sodium bicarbonate exhibited less evidence of tissue breakdown than the samples stored without preservatives or with only one of those preservatives. In compression testing, samples tested immediately after harvest were significantly stiffer than samples tested after 24 h of storage at 10°C in ACSF (difference: -0.27 N/mm, 95% confidence interval (CI): -0.50, -0.05) or ACSF with antibiotics/antifungal agents (difference: -0.32 N/mm, 95% CI: -0.59, -0.04), controlling for harvest lobe. In contrast, the stiffness of samples tested after storage in either solution containing sodium bicarbonate was not significantly different from the stiffness of samples tested at harvest. There was no significant overall difference in the mean tissue stiffness between samples from the frontal and parietal lobes, controlling for storage solution. Given the importance of PMHS studies to brain injury research, any strategy that shows promise for helping to maintain in vivo brain material properties has the potential to improve understanding of brain injury mechanisms and tolerance to head injury and warrants further investigation. These pilot study results suggest that sodium bicarbonate has the potential to reduce the deterioration of brain tissue in biomechanical testing. The results motivate further evaluation of sodium bicarbonate as a preservative for biomechanical testing using additional test subjects, more comprehensive material testing, and evaluation under a broader set of test conditions including in whole-head testing. The effect of antibiotics and antifungal agents on brain tissue stiffness was minimal but may have been limited by the cold storage conditions in this study. Further exploration of the potential for microbial agents to preserve tissue postmortem would benefit from evaluation of the effects of storage temperature.

Disentangling the drivers of urban bird diversity in the non-breeding season: A general synthesis.

Current knowledge about the impacts of urbanisation on bird assemblages is based on evidence from studies partly or wholly undertaken in the breeding season. In comparison, the non-breeding season remains little studied, despite the fact that winter conditions at higher latitudes are changing more rapidly than other seasons. During the non-breeding season, cities may attract or retain bird species because they offer milder conditions or better feeding opportunities than surrounding habitats. However, the range of climatic, ecological and anthropogenic mechanisms shaping different facets of urban bird diversity in the non-breeding season are poorly understood. We explored these mechanisms using structural equation modelling to assess how urbanisation affects the taxonomic, phylogenetic and functional diversity of avian assemblages sampled worldwide in the non-breeding season. We found that minimum temperature, elevation, urban area and city age played a critical role in determining taxonomic diversity while a range of factors-including productivity, precipitation, elevation, distance to coasts and rivers, socio-economic (as a proxy of human facilitation) and road density-each contributed to patterns of phylogenetic and functional diversity. The structure and function of urban bird assemblages appear to be predominantly shaped by temperature, productivity and city age, with effects of these factors differing across seasons. Our results underline the importance of considering multiple hypotheses, including seasonal effects, when evaluating the impacts of urbanisation on biodiversity.

Anxiolytic effect of alamandine in male transgenic rats with low brain angiotensinogen is dependent on activation of MrgD receptors.

Alamandine is a peptide hormone belonging to the renin-angiotensin system (RAS). It acts through the Mas-related G-protein coupled receptor type D, MrgD, which is expressed in different tissues, including the brain. In the present study, we hypothesize that a lack of alamandine, through MrgD, could cause the anxiety-like behavior in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)680]. Adult male transgenic rats exhibited a significant increase in the latency to feeding time in the novelty suppressed feeding test and a decrease in the percentage of time and entries in the open arms in the elevated plus maze. These effects were reversed by intracerebroventricular infusion of alamandine. Pretreatment with D-Pro7-Ang-(1-7), a Mas and MrgD receptor antagonist, prevented the anxiolytic effects induced by this peptide. However, its effects were not altered by the selective Mas receptor antagonist, A779. In conclusion, our data indicates that alamandine, through MrgD, attenuates anxiety-like behavior in male TGR(ASrAOGEN)680, which reinforces the importance of the counter-regulatory RAS axis as promising target for the treatment of neuropsychiatric disorders.

Know-how of holding a Bioinformatics competition: Structure, model, overview, and perspectives.

The article presents a framework for a Bioinformatics competition that focuses on 4 key aspects: structure, model, overview, and perspectives. Structure represents the organizational framework employed to coordinate the main tasks involved in the competition. Model showcases the competition design, which encompasses 3 phases. Overview presents our case study, the League of Brazilian Bioinformatics (LBB) 2nd Edition. Finally, the section on perspectives provides a brief discussion of the LBB 2nd Edition, along with insights and feedback from participants. LBB is a biannual team competition launched in 2019 to promote the ongoing training of human resources in Bioinformatics and Computational Biology in Brazil. LBB aims to stimulate ongoing training in Bioinformatics by encouraging participation in competitions, promoting the organization of future Bioinformatics competitions, and fostering the integration of the Bioinformatics and Computational Biology community in the country, as well as collaboration among participants. The LBB 2nd Edition was launched in 2021 and featured 251 competitors forming 91 teams. Knowledge competitions promote learning, collaboration, and innovation, which are crucial for advancing scientific knowledge and solving real-world problems. In summary, this article serves as a valuable resource for individuals and organizations interested in developing knowledge competitions, offering a model based on our experience with LBB to benefit all levels of Bioinformatics trainees.

Fire effects on pollination and plant reproduction: a quantitative review.

BACKGROUND AND AIMS: Fire may favour plant flowering by opening the vegetation and increasing abiotic resource availability. Increased floral display size can attract more pollinators and increase the absolute fruit and seed production immediately after the fire. However, anthropogenic increases in fire frequency may alter these responses. We aim to assess the effects of fire on pollination and reproductive success of plants at the global scale. METHODS: We performed a systematic literature review and meta-analyses to examine overall fire effects as well as different fire parameters on pollination and on plant reproduction. We also explored to what extent the responses vary among pollinators, pollination vectors, plant regeneration strategies, compatibility systems, vegetation types and biomes. KEY RESULTS: Most studies were conducted in fire-prone ecosystems. Overall, single fires increased pollination and plant reproduction but this effect was overridden by recurrent fires. Floral visitation rates of pollinators were enhanced immediately following a wildfire, and especially in bee-pollinated plants. Fire increased the absolute production of fruits or seeds but not the fruit or seed set. The reproductive benefits were mostly observed in wind-pollinated (graminoids), herbaceous and resprouter species. Finally, fire effects on pollination were positively correlated with fire effects on plant reproductive success. CONCLUSIONS: Fire has a central role in pollination and plant sexual reproduction in fire-prone ecosystems. The increase in the absolute production of fruits and seeds suggests that fire benefits on plant reproduction are likely driven by increased abiotic resources and the consequent floral display size. However, reproduction efficiency, as measured by fruit or seed set, does not increase with fire. In contrast, when assessed on the same plant simultaneously, fire effects on pollination are translated into reproduction. Increased fire frequency due to anthropogenic changes can alter the nature of the response to fire.

Anthropogenic land-use change decreases pollination and male and female fitness in terrestrial flowering plants.

BACKGROUND AND AIMS: The majority of the earth's land area is currently occupied by humans. Measuring how terrestrial plants reproduce in these pervasive environments is essential for understanding their long-term viability and their ability to adapt to changing environments. METHODS: We conducted hierarchical and phylogenetically-independent meta-analyses to assess the overall effects of anthropogenic land-use changes on pollination, and male and female fitness in terrestrial plants. KEY RESULTS: We found negative global effects of land use change (i.e., mainly habitat loss and fragmentation) on pollination and on female and male fitness of terrestrial flowering plants. Negative effects were stronger in plants with self-incompatibility (SI) systems and pollinated by invertebrates, regardless of life form and sexual expression. Pollination and female fitness of pollination generalist and specialist plants were similarly negatively affected by land-use change, whereas male fitness of specialist plants showed no effects. CONCLUSIONS: Our findings indicate that angiosperm populations remaining in fragmented habitats negatively affect pollination, and female and male fitness, which will likely decrease the recruitment, survival, and long-term viability of plant populations remaining in fragmented landscapes. We underline the main current gaps of knowledge for future research agendas and call out not only for a decrease in the current rates of land-use changes across the world but also to embark on active restoration efforts to increase the area and connectivity of remaining natural habitats.

Experimental and Theoretical Study on Electron Ionization and Fragmentation of Propylene Oxide─the First Chiral Molecule Detected in the Interstellar Medium.

Propylene oxide, CH3CHOCH2, is the first chiral molecule detected in space and the third C3 oxide detected toward the Sagittarius B2 (Sgr B2 (N)) molecular cloud, the others being propanal, CH3CH2CHO, and acetone, (CH3)2CO. With homochirality being ubiquitous in the building blocks of living matter, the formation and decay paths of propylene oxide in space are of specific interest. Motivated by the significant role of photo- and secondary electrons in astrochemistry, we have studied electron ionization and fragmentation of propylene oxide. Ion appearance energies are determined and compared to threshold values for the respective processes calculated at the G4MP2 level of theory, and potential reaction pathways are computed at the DFT level of theory. Electron ionization is found to destabilize propylene oxide, leading to barrierless opening of the C1-C2 bond of the epoxy ring, hydrogen transfer, and fragmentation over the methyl vinyl ether or rupture of the C2-O bond of the epoxy ring and fragmentation of the allyl alcohol cation as an intermediate, rather than direct bond ruptures.

Racial and ethnic disparities in motor vehicle crash-related outcomes in North Carolina surrounding the COVID-19 pandemic.

Long-term impacts of the COVID-19 pandemic on racial and ethnic disparities in motor vehicle crash (MVC) injuries and death are poorly understood. This study aimed to characterize trends and investigate the heterogeneity of MVC-related disparities in North Carolina across several data sources. Crash reports, emergency department visit records, and death certificates from 2018 to 2021 were used to calculate monthly population-rates of MVC-related public health outcomes. We estimated trendlines using joinpoint regression and compared outcomes across racial and ethnic classifications. MVC and MVC-related injury rates declined in conjunction with NC's stay-at-home order, while rates of severe outcomes remained unimpacted. By December 2021 rates of MVC-related outcomes met or exceeded pre-pandemic levels, with the highest rates observed among non-Hispanic Black individuals. Racial and ethnic disparities in MVC-related outcomes remained prevalent throughout the COVID-19 pandemic. These results highlight the importance of a holistic approach to traffic injury surveillance when assessing the impact of MVCs.