bluuebunny/crossref_metadata_2025 huggingface dataset:
Aerosolized ⍺1adrenoreceptor antagonism does not affect experimentally induced lung fibrosis in animal models
Authors: Ghincea Alexander , Perry Carrighan , Liu Angela , McGovern John , Yu Sheeline , Peng Xue-Yan , Ishikawa Genta , Barnthaler Thomas , Herzog Erica L. , Sun Huanxing
Published: December 2023

Pulmonary Fibrosis is a progressive and incurable condition that complicates many disease states. Adrenergic hyperinnervation and accumulation of fibroblasts expressing ⍺1- adrenoreceptors have been implicated in this process. Previous studies have demonstrated that systemic treatment with an ⍺1-adrenoreceptors antagonist attenuates fibrotic endpoints in lung fibrosis models. In an attempt to develop a lung targeted therapy, we determined whether ⍺1- adrenoreceptors antagonism delivered via inhaled administration of terazosin exerts antifibrotic benefits in experimentally induced lung fibrosis. C57/BL6 mice treated with bleomycin, or a doxycycline inducible line of transgenic mice with lung specific overexpression of the bioactive form of the human TGFβ1 (TGFβ1-Tg+model), received nebulized terazosin at varying doses on a therapeutic schedule following the induction of fibrosis and were sacrificed at 21 days. Airway inflammation, fibrotic endpoints, and lung function were evaluated. ⍺1-adrenoreceptors antagonism delivered via this method did not impact airway inflammation as indicated by bronchoalveolar lavage cell counts, and there was no significant difference observed in soluble collagen content. There was similarly no significant difference in respiratory mechanics with terazosin administration. These data show that inhaled delivery of the ⍺1-adrenoreceptors antagonist terazosin by this method is ineffective at treating fibrosis in these models and suggest that alternative dosing schedules or delivery methods may be more fruitful avenues of investigation. Further exploration of these findings may provide new therapeutic options and illuminate mechanisms through which adrenergic innervation and ⍺1-adrenoreceptors mediate fibrosis in the adult mammalian lung.
Transgenic mice overexpressing Pitx2 in the atria develop tachycardia-bradycardia syndrome
Authors: Baba Shunsuke , Shinjo Satoko , Seya Daiki , Bochimoto Hiroki , Akaike Toru , Nakano Atsushi , Minamisawa Susumu
Published: December 2023

Sinoatrial node (SAN) dysfunction often accompanies supraventricular tachyarrhythmias such as atrial fibrillation (AF), which is referred to as tachycardia-bradycardia syndrome (TBS). Although there have been many studies on electrical remodeling in TBS, the regulatory mechanisms that cause electrical remodeling in the SAN and atrial muscles by chronic bradycardia or tachycardia have not yet been fully investigated. Here we hypothesized that Pitx2c, a transcription factor that plays a central role in the late aspects of left-right asymmetric morphogenesis, regulates an interrelationship between the SAN and the atrial muscles and is involved in TBS-like pathology. To test this hypothesis, we generated transgenic mice overexpressingPitx2cspecifically in the atria (OE mice). Although Pitx2c is normally expressed only in left atria, the expression levels of Pitx2c protein in the right atria were significantly increased to similar levels of those in the left atria of non-transgenic control mice (WT). We found that the heart rate of OE mice was significantly variable although the average heart rate was similar between WT and OE mice. Electrophysiological examination showed that OE mice exhibited prolonged SAN recovery time and higher AF inducibility. In addition, recording of the atrial monophasic action potential duration using a Langendorff perfusion system demonstrated shorter action potential duration in OE atria. Histological analysis revealed that SAN-specific ion channel HCN4-positive cells were hardly detected in the SAN of OE mice, along with ectopic expression in the right atria. Furthermore, transcription factors associated with sinus node formation were down-regulated in the right atria of OE mice. Therefore, SAN dysfunction by Pitx2 dysregulation predisposed OE mice to a TBS-like phenotype. We conclude that Pitx2c is a key regulator that defines SAN function in the atria.
Accumulation of per- and polyfluoroalkyl substances (PFAS) in a terrestrial food web
Authors: Ecke Frauke , Skrobonja Alexandra , Malmsten Jonas , Ahrens Lutz
Published: December 2023

Per– and polyfluoroalkyl substances (PFAS) are synthetic organofluorine chemical compounds that are broadly used in amongst others aqueous firefighting foam, cosmetics, textiles, carpets, coatings, plastics, and ski wax. Their chemical properties make them persistent organic pollutants that are potential bioaccumulative and toxic. Most studies on PFAS have been performed in groundwater, surface water and aquatic biota. Our knowledge on the terrestrial fate of PFAS is therefore limited.

We sampled soil, berries, mushrooms, and wildlife on the island Frösön, central Sweden, in 2021 and 2022, to study the fate of 22 PFAS in a terrestrial food web. Groundwater, surface water and fish on Frösön have previously shown high PFAS concentrations. Soil, berries, and mushrooms were also concurrently sampled in a reference area in northern Sweden.

Overall, concentrations of the sum of PFAS were low in berries and mushrooms. In moose (Alces alces), roedeer (Capreolus capreolus), and bank vole (Myodes glareolus), concentrations were highest in liver. The maximum levels for PFOS in offal as set by the European Commission (50 ng/g ww) were exceeded in the liver of two of 10 roedeer. Bank voles (n= 12 pools) that were sampled in the vicinity of a firefighting training site showed partly extreme concentrations with none of the samples having liver concentrations <474 ng/g ww (maximum 11,600 ng/g ww). Bioaccumulation factors (BAFs) for multiple PFAS in bank voles were higher for the studied mushrooms and soil compared to studied berries and were >100 for 27 out of 265 calculated BAFs. BAFs in the ungulate species were generally lower than those for bank voles but were >1 for several PFAS from the mushroom.

The exact origin of PFAS in bank voles could not be identified in our study, but the BAFs are supported by the feeding and behaviour ecology of bank voles, i.e., there is likely bioaccumulation and biomagnification from soil and mushrooms to bank voles. The measured PFAS concentrations and BAFs, especially those in bank voles are worrying from an ecosystem and One Health perspective considering that voles are staple food for multiple predators.
Protein search processes mediated by chromatin topology
Authors: Dutta Shuvadip , Adarshkrishnan R. , Padinhateeri Ranjith , Mitra Mithun K.
Published: December 2023

We investigate the role of compaction of chromatin domains in modulating search kinetics of proteins. Collapsed conformations of chromatin, characterised by long loops which bring distant regions of the genome into contact, and manifested structurally as Topologically Associated Domains (TADs) affect search kinetics of DNA associated transcription factors and other proteins. In this study, we investigate the role of the compactness of chromatin on the dynamics of proteins using a minimal model. Using analytical theory and simulations, we show that an optimal compaction exists for which the residence time of proteins on a chromatin-like polymer backbone is minimum. We show that while bulk diffusion is an advantageous search strategy for extended polymers, for highly folded polymer domains, intersegmental transfers allow optimal search. We extend these results to more detailed polymer models - using the Freely Rotating Chain model, a Lennard-Jones bead-spring polymer model, which approximates chromatin behavior. We show that our results continue to hold for these polymer models, with a minimum residence time at an optimum polymer compaction. Finally, we also analyse the dynamics of proteins on networks generated using experimental chromatin conformation data from 8355 TADs extracted from human chromosomes. Our analysis suggests that TADs exist near this zone of optimality, indicating that chromatin conformations can play a crucial role in modulating protein search strategies.
Selection bias in reporting of median waiting times in organ transplantation
Authors: Schwab Simon , Elmer Andreas , Sidler Daniel , Straumann Lisa , Stürzinger Ueli , Immer Franz
Published: December 2023

Median organ waiting times published by transplant organizations around the world may be biased when death or censoring is disregarded. This can lead to too optimistic waiting times for all organ waiting lists, but most strikingly in kidney transplantation, and as a consequence, may deceive patients on the waiting list, transplant physicians, and healthcare policy maker. In this cohort study of all trans-plant candidates in Switzerland, we use competing risk multistate models for the analysis of time-to-event data of the organ waiting list. The resulting cumulative incidences are probabilities for transplantation or death by a given time are a more accurate description of the events occurring on the waiting list. In accordance with the concept of median survival time in survival analysis in clinical trials, we suggest the median time to transplantation (MTT), the waiting time duration at which the transplant probability is 0.50, as a measure of average waiting time.

Summary points

Transplant candidates on the waiting list, healthcare professionals and policy makers may be interested in the accurate assessment of the probabilities of transplantation and undesirable events.

The median waiting time based on the median calculated across the waiting durations of transplanted individuals may be misleading due to selection bias, ignoring death and censored observations.

Competing risk multistate models based on the Aalen-Johansen estimator are effective in assessing the waiting list with multiple competing events.

From the cumulative incidence curve the probabilities of transplantation or death can be determined for any time period.

We propose to report both the median time to transplantation (MTT), i.e. the waiting time duration at which the transplant probably is 50%, as well as the probabilities of other competing events during MTT.

National transplant organizations around the world may consider changing their reporting policy regarding waiting list statistics.
Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals
Authors: Phillips Mary L. , Urban Nicolai T. , Salemi Taddeo , Dong Zhe , Yasuda Ryohei
Published: December 2023

Head-mounted miniscopes have allowed for functional fluorescence imaging in freely moving animals. However, current capabilities of state-of-the-art technology can record only up to two, spectrally distinct fluorophores. This severely limits the number of cell types identifiable in a functional imaging experiment. Here we present a pipeline that enables the distinction of nine neuronal subtypes from regions defined by behaviorally relevant cells duringin vivoGCaMP imaging. These subtypes are identified utilizing unique fluorophores that are co-expressed with GCaMP, unmixed by spectral imaging on a confocal microscope and co-registering these spectral fingerprints with functional data obtained on miniaturized microscopes. This method facilitates detailed analyses of circuit-level encoding of behavior.
Rate Dependent Cochlear Outer Hair Cell Force Generation: Models and Parameter Estimation
Authors: Cai Wen , Grosh Karl
Published: December 2023

The outer hair cells (OHCs) of the mammalian cochlea are the mediators of an active, nonlinear electromechanical process necessary for sensitive, frequency specific hearing. The membrane protein prestin conveys to the OHC a piezoelectric-like behavior hypothesized to actuate a high frequency, cycle-by-cycle conversion of electrical to mechanical energy to boost cochlear responses to low-level sound. This hypothesis has been debated for decades, and we address two key remaining issues: the influence of the rate dependence of conformal changes in prestin and the OHC transmembrane impedance. We develop a theoretical electromechanical model of the OHC that explicitly includes rate dependence of conformal transitions, viscoelasticity, and piezoelectricity. Using this theory, we show the influence of rate dependence and viscoelasticity on electromechanical force generation. Further, we stress the importance of using the correct mechanical boundary conditions when estimating the transmembrane capacitance. Finally, a set of experiments is described to uniquely estimate the constitutive properties of the OHC from whole-cell measurements.
Harnessing light-activated gallium porphyrins to combat intracellularStaphylococcus aureusin dermatitis: Insights from a simplified model
Authors: Szymczak Klaudia , Rychłowski Michał , Zhang Lei , Nakonieczna Joanna
Published: December 2023

Staphylococcus aureuscan survive inside nonprofessional phagocytes such as keratinocytes, demonstrating a novel strategy for evading antibiotic pressure. When antibiotic treatment ends, reinfection with staphylococci begins from the intracellular inoculum. This phenomenon is responsible for recurrent infections. The development of new antibacterial methods that can eliminate intracellular bacteria, including those with a multidrug-resistant phenotype, is necessary. In this study, we characterized and used a model of keratinocytes (both wild type and mutants with reduced filaggrin expression) infected with methicillin-resistantS. aureus(MRSA) to verify the possibility of using light-activated compounds, exemplified here by heme-mimetic gallium (III) porphyrin (Ga3+CHP) and visible light, an approach known as antimicrobial photodynamic inactivation (aPDI), to eliminate intracellular MRSA. We observed that Ga3+CHP accumulated more in infected cells than in uninfected cells. Moreover, Ga3+CHP accumulated in cells that harbored intracellularS. aureus. Using flow cytometry and fluorescence microscopy, we found that intracellular MRSA and Ga3+CHP mainly colocalized in lysosomal structures, and we showed that under the influence of aPDI, MRSA exhibited reduced adhesion to host cells and a significantly reduced (by 70%) GFP signal originating from intracellular bacteria. Moreover, the use of light-activated Ga3+CHP resulted in a significant reduction in the number of extracellular bacteria in the infection system, lowering the potential for further infection of host cells. For the first time, we used the infectious model to analyze the toxicity of aPDI in real time, showing that this approach is not significantly cyto-or phototoxic.

Author Summary

Staphylococcus aureusis a highly virulent pathogen that is responsible for approximately 80% of all skin infections. During antibiotic treatment, one of the defense mechanisms ofS. aureusis the invasion of keratinocytes. Intracellular bacteria are not accessible to antibiotics, which poorly penetrate the interior of host cells. Consequently, such bacteria contribute to recurrent infections. In our study, we proposed using a combination of a light-activated porphyrin compound loaded with gallium ions, Ga3+CHP, and visible light as a strategy to eliminate intracellular staphylococci. We demonstrated that the tested compound colocalized with the pathogen in the infected cells, which was an essential condition for the effective elimination of intracellular bacteria. We showed that the proposed approach effectively reduced the infection of keratinocytes with methicillin-resistantS. aureus(MRSA), as well as its adhesion to host cells, while maintaining host cells. The results presented here provide a basis for developing an effective therapy against staphylococci.
Knowing what you don’t know: Estimating the uncertainty of feedforward and feedback inputs with prediction-error circuits
Authors: Hertäg Loreen , Wilmes Katharina A. , Clopath Claudia
Published: December 2023

At any moment, our brains receive a stream of sensory stimuli arising from the world we interact with. Simultaneously, neural circuits are shaped by feedback signals carrying predictions about the same inputs we experience. Those feedforward and feedback inputs often do not perfectly match. Thus, our brains have the challenging task of integrating these conflicting streams of information according to their reliabilities. However, how neural circuits keep track of both the stimulus and prediction uncertainty is not well understood. Here, we propose a network model whose core is a hierarchical prediction-error circuit. We show that our network can estimate the variance of the sensory stimuli and the uncertainty of the prediction using the activity of negative and positive prediction-error neurons. In line with previous hypotheses, we demonstrate that neural circuits rely strongly on feedback predictions if the perceived stimuli are noisy and the underlying generative process, that is, the environment is stable. Moreover, we show that predictions modulate neural activity at the onset of a new stimulus, even if this sensory information is reliable. In our network, the uncertainty estimation, and, hence, how much we rely on predictions, can be influenced by perturbing the intricate interplay of different inhibitory interneurons. We, therefore, investigate the contribution of those inhibitory interneurons to the weighting of feedforward and feedback inputs. Finally, we show that our network can be linked to biased perception and unravel how stimulus and prediction uncertainty contribute to the contraction bias.
Response diversity in the context of multifarious environmental change
Authors: Polazzo Francesco , Limberger Romana , Pennekamp Frank , Ross Samuel R. P.-J. , Simpson Gavin L. , Petchey Owen L.
Published: December 2023

1.

Response diversity represents the inter- and intraspecific trait variation in organismal responses to the environment. Assemblages composed of organisms displaying large variation in their response to the environment (that is, having high response diversity) are expected to have higher temporal stability of aggregate community and ecosystem properties such as ecosystem functioning (i.e., an insurance effect). Yet, response diversity is not commonly measured in empirical studies, and when it is measured, this is done in different ways. Moreover, most proposed measures of response diversity concern situations with only one driver of environmental change. Thus far, no specific approach exists to measure response diversity in the context of multiple simultaneously changing (multifarious) environmental drivers. Here, we propose a new method to empirically quantify response diversity in the context of multifarious environmental change. First, we illustrate this method using simulated data. Next, we reveal the role of the direction of environmental change in shaping response diversity when multiple drivers of environmental change fluctuate over time. We show that, when the direction of the environmental change is unknown (that is, there is no information ora prioriexpectation about how an environmental condition has changed or will change in future), we can quantify thepotentialresponse diversity for a given community under any possible future environmental change scenario. That is, we can estimate the potential response capacity of a system under a range of extreme or realistic environmental changes, capturing its complete insurance capacity, with utility for predicting future responses to even multifarious environmental change. Finally, we investigate the drivers of response diversity in a multifarious environmental change context, showing how response diversity depends on: 1) the diversity of species responses to each environmental variable considered, 2) the relative effect of each environmental variable on species’ performance, 3) the correlation between the diversity in species’ responses to different environmental variables, and 4) the mean temporal value of the environmental variable. In doing so, we take an important step towards understanding the insurance capacity of ecological communities exposed to multifarious environmental change.
A barley MLA receptor is targeted by a non-ribosomal peptide effector of the necrotrophic spot blotch fungus for disease susceptibility
Authors: Leng Yueqiang , Kümmel Florian , Zhao Mingxia , Molnár István , Doležel Jaroslav , Logemann Elke , Köchner Petra , Xi Pinggen , Yang Shengming , Moscou Matthew J. , Fiedler Jason D. , Du Yang , Steuernagel Burkhard , Meinhardt Steven , Steffenson Brian J. , Schulze-Lefert Paul , Zhong Shaobin
Published: December 2023

The evolutionary history of plant interactions with necrotrophic pathogens that feed on dying host cells and their virulence mechanisms remains fragmentary. We have isolated the barley geneScs6, which is required for the necrotrophic fungusBipolaris sorokinianaisolate ND90Pr to cause spot blotch disease.Scs6is located at the disease resistance gene locusMildew locus a(Mla) and encodes an intracellular nucleotide-binding leucine-rich repeat receptor (NLR). In transgenic barley,Scs6is sufficient to confer susceptibility to ND90Pr in accessions naturally lacking the receptor, resulting in infection-associated host cell death. Expression ofScs6in evolutionarily distantNicotiana benthamianareconstitutes a cell death response to an uncharacterized non-ribosomal peptide effector produced by ND90Pr-specific non-ribosomal peptide synthetases (NRPSs) encoded at theVHv1virulence locus. Our data suggest that the heat-resistant effector directly activates the SCS6 receptor.Scs6is an allelic variant of functionally diversifiedMlaresistance genes each conferring strain-specific immunity to barley powdery mildew isolates with a matching proteinaceous pathogen effector. Domain swaps between MLA and SCS6 NLRs and expression of the resulting hybrid proteins inN. benthamianareveal that the SCS6 leucine-rich repeat domain is a specificity determinant for the NRPS-derived effector to activate the receptor.Scs6evolved after the divergence of barley from wheat and is maintained in several wild barley populations with an incidence of 8%, suggesting a beneficial function for the host. Evolution of thebona fideimmune receptor SCS6 targeted by the NRPS-derived effector was key for the emergence of strain-specific spot blotch disease in domesticated barley.
The prefoldin-like protein AtURI exhibits characteristics of instrinsically disordered proteins
Authors: Gómez-Mínguez Yaiza , Palacios-Abella Alberto , Costigliolo-Rojas Cecilia , Barber Mariana , Hernández-Villa Laura , Úrbez Cristina , Alabadí David
Published: December 2023

The prefoldin-like protein UNCONVENTIONAL PREFOLDIN RPB5 INTERACTOR (URI) participates in diverse cellular functions, including protein homeostasis, transcription, translation, and signal transduction. Thus, URI is a highly versatile protein, although the molecular basis of this versatility remains unknown. In this work, we show thatArabidopsis thaliana(Arabidopsis) URI (AtURI) possesses a large intrinsically disordered region (IDR) spanning most of the C-terminal part of the protein, a feature conserved in yeast and human orthologs. Our findings reveal two key characteristics of disordered proteins in AtURI: promiscuity in interacting with partners and protein instability. We propose that these two features contribute to providing AtURI with functional versatility.
In vitro, Evaluation for Consumption Ratio for certain plants and Toxicity and Biochemical Parameters of some Insecticides onEobania vermiculata
Authors: Bashandy A.S.
Published: December 2023

Land snails are one of the most harmful agricultural pests to crops, causing economic devastation to a variety of plants. The purpose of this study is to determine the optimal food preference of land snails,Eobania vermiculata, and evaluate the mortality and biochemical activity of five pesticides using a leaf dipping technique against them in vitro. The findings revealed that in “no-choice feeding,” it was observed that land snails consumed a significantly higher amount of Cos lettuce leaves compared to leaves from other plants. However, chicory leaves were found to be the least preferred by the snails. On the other hand, “free choice feeding,” revealed that land snails showed a preference for Cos lettuce and cabbage leaves, which were consumed more frequently. Conversely, Komatsuna leaves had the lowest acceptance among the snails. Also, the results indicated the mortality rate increased with an increased concentration during exposure time. After three days, the lower concentrations of Sulfoxaflor and Fipronil caused a high mortality percentage in the animals compared with other pesticides, respectively. Moreover, after one month of exposure to 1000 ppm/100 ml, Chlorantraniliprole caused 46.43% mortality. But Sulfoxaflor and Fipronil exhibited (42.86%) mortality. They achieved LC50(1010.48, 2501.93, and 1444.66 ppm/100ml), against tested snails, respectively. Nevertheless, Spinetoram and Spirotetramat caused a lower mortality rate. During seven days of exposure to LC25for five pesticides by leaf dipping on E. vermiculata. They impacted some of the biochemical activity of two enzymes: alanine amino transaminase (ALT), aspartate amino transaminase (AST), total proteins (LP), and the lipid profile of E. vermiculata. The results showed that the activities of ALT, AST, and Triglycerides rose by 50%, with 108 u/l, 407 u/l, and 2064 mg/dl, respectively. Compared to the control and other compounds, Spirotetramat raised total cholesterol by 33 mg/dl. ALT and AST activity and Triglycerides lowered following Spinetoram and Fipronil treatment at 13 u/l, 32u/l, and (4 mg/dl) of TL, respectively. However, no substantial treatments or controls that influence TP, (Total cholesterol and LDH) or LP levels after the exposure period are available. Ultimately, the results indicated that both Cos lettuce and cabbage leaves were the preferred food choices for land snails, as observed in both feeding methods. However, it is important to note that these findings also highlight the need to consider the impact of insecticides on land snails. Incorporating these insecticides into a comprehensive management strategy to mitigate any negative effects of land snails while ensuring the overall well-being of the environment.
NeMu: A Comprehensive Pipeline for Accurate Reconstruction of Neutral Mutation Spectra from Evolutionary Data
Authors: Efimenko Bogdan , Popadin Konstantin , Gunbin Konstantin
Published: December 2023

One of the most important characteristics of each contemporary model of molecular evolution is the assumption that mutations occur in a constant manner; however, in the real world, the mutations are determined by the combination of the effects of DNA replication and repair. This affects the nucleotide composition of the genome and guides not just neutral but adaptive evolution1. Mutation accumulation experiments are the de facto standard for the neutral mutation spectra estimation. However, recent studies have demonstrated that the mutation fraction under selection pressure is significantly underestimated in mutation accumulation experiments, and, therefore the precise extraction of neutral mutation spectra from mutation accumulation experiments is not trivial2. To unravel the neutral mutation spectra, it is very important to analyze all the mutations available in depth, based on the evolutionary timescale, taking into consideration all the existing knowledge. In order to facilitate this analysis, we have created a novel pipeline, called NeMu (https://biopipelines.kantiana.ru/nemu/).
A complex regulatory cascade involving quorum sensing regulates siderophore-mediated iron homeostasis inChromobacterium violaceum
Authors: Batista Bianca B. , de Lima Vinicius M. , Picinato Beatriz A. , Koide Tie , da Silva Neto José F.
Published: December 2023

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogenChromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos on PSA-CAS plates identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis inC. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression ofvioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations incviRand null mutation ofcviIandcviRled to an increase of siderophore halos. RNA-seq of thecviIandcviRmutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis inC. violaceum.

Importance

The iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems inChromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor ofvioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of avitRmutant were rescued in avitRvioSmutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores inC. violaceum.
Microbiome depletion and recovery in the sea anemone,Exaiptasia diaphana, following antibiotic exposure
Authors: MacVittie Sophie , Doroodian Saam , Alberto Aaron , Sogin Maggie
Published: December 2023

Microbial species that comprise host-associated microbiomes play an essential role in maintaining and mediating the health of plants and animals. While defining the role of individual or even complex communities is important towards quantifying the effect of the microbiome on host health, it is often challenging to develop causal studies that link microbial populations to changes in host fitness. Here, we investigated the impacts of reduced microbial load following antibiotic exposure on the fitness of the anemone,Exaiptasia diaphanaand subsequent recovery of the host’s microbiome. Anemones were exposed to two different types of antibiotic solutions for three weeks and subsequently held in sterilized seawater for a subsequent three-week recovery period. Our results revealed that both antibiotic treatments reduced the overall microbial load during and up to one week post treatment. The observed reduction in microbial load was coupled to reduced anemone biomass, halted asexual reproduction rates, and for one of the antibiotic treatments, the partial removal of the anemone’s algal symbiont. Finally, our amplicon sequencing results of the 16S rRNA gene revealed that anemone bacterial composition only shifted in treated individuals during the recovery phase of the experiment, where we also observed a significant reduction in the overall diversity of the microbial community. Our work implies that theE. diaphana’smicrobiome contributes to host fitness and that the recovery of the of the host’s microbiome following disturbance with antibiotics leads to a reduced, but stable microbial state.

Importance

Exaiptasia diaphanais an emerging model used to define the cellular and molecular mechanisms of coral-algal symbioses.E. diaphanaalso houses a diverse microbiome, consisting of hundreds of microbial partners with undefined function. Here, we applied antibiotics to quantify the impact of microbiome removal on host fitness as well as define trajectories in microbiome recovery following disturbance. We showed that reduction of the microbiome leads to negative impacts on host fitness, and that the microbiome does not recover to its original composition while held under aseptic conditions. Rather the microbiome becomes less diverse, but more consistent across individuals. Our work is important because it suggests that anemone microbiomes play a role in maintaining host fitness, that they are susceptible to disturbance events, and it is possible to generate gnotobiotic individuals that can be leveraged in microbiome manipulation studies to investigate the role of individual species on host health.
Physiological and transcriptomic response to methyl-coenzyme M reductase limitation inMethanosarcina acetivorans
Authors: Chadwick Grayson L. , Dury Gavin A. , Nayak Dipti D.
Published: December 2023

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural studies have generated detailed insights into MCR functionin vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this statement has not been categorically tested. Here, to gain a more direct understanding of MCR’s control on the growth ofMethanosarcina acetivorans,we generate a strain with an induciblemcroperon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis ofM. acetivoransexperiencing MCR-limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to a strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, themcroperon does not seem to be transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors like anabolic reactions might be rate-limiting for methanogenic growth.

Importance

Methane is a potent greenhouse gas that has contributed toca.25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. In methanogens, methyl-coenzyme M reductase (MCR) catalyzes methane formation. Even though MCR comprisesca.10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. Here, we show thatMethanosarcina acetivoransgrown under standard laboratory conditions produces more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher throughput manner than isolation and biochemical characterization of pure protein.
Molecular definition of the BAK:VDAC2 interaction as a target to manipulate apoptosis
Authors: Yuan Zheng , van Delft Mark , Li Mark , Sumardy Fransisca , Smith Brian J , Huang David C. S. , Lessene Guillaume , Khakam Yelena , Jin Ruitao , He Sitong , Smith Nicholas A , Birkinshaw Richard W , Czabotar Peter E , Dewson Grant
Published: December 2023

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, whilst interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK, and inhibited BAK apoptotic activity. Thus, this VDAC2–BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease, or to promote BAK-mediated apoptosis for cancer therapy.
Spatial Transcriptomic Characterization of Novel Pathologic Niches in IPF
Authors: Mayr Christoph H. , Santacruz Diana , Jarosch Sebastian , Lempp Charlotte , Neubert Lavinia , Rath Berenice , Kamp Jan C. , Jonigk Danny , Kühnel Mark , Schlueter Holger , Doerr Jonas , Dick Alec , Ramirez Fidel , Thomas Matthew J.
Published: December 2023

An unmet medical need persists in Idiopathic Pulmonary fibrosis (IPF), for which treatments additional to anti-fibrotic therapy are needed. Single cell RNA sequencing (scRNA-seq) has advanced our understanding of IPF with cell type-specific insights but lacks cellular tissue context. Spatial transcriptomics addresses this by providing spatially resolved gene expression, enabling gene and cell type localization within the tissue environment. We profiled IPF and control patient lung tissue sections using spatial transcriptomics and combined the data with an atlas of integrated IPF scRNA-seq datasets. Through computational analysis, we identified three disease-associated pathologic niches with unique cellular composition / localization and analyzed their cell-cell communication. We identified the Fibrotic niche, comprising Myofibroblasts and Aberrant Basaloid cells, preferentially located around airways and close to the Airway Macrophage niche in the lumen, containing SPP1+ Macrophages. We also identified the Immune niche, distinct foci of lymphoid cells in fibrotic tissue, surrounded by remodeled endothelial vessels.

TEASER

Spatial transcriptomics localizes genes and cell types in the tissue and identifies pathological cellular niches in IPF and control lungs.
A translation-independent directed evolution strategy to engineer aminoacyl-tRNA synthetases
Authors: Soni Chintan , Prywes Noam , Hall Matthew , Savage David F. , Schepartz Alanna , Chatterjee Abhishek
Published: December 2023

Using directed evolution, engineered aminoacyl-tRNA synthetases (aaRS) have been developed that enable co-translational incorporation of numerous noncanonical amino acids (ncAAs) into proteins in living cells. Until now, the selection of such novel aaRS mutants has relied on coupling their activity to the expression of a reporter protein with a selectable phenotype. However, such translation-dependent selection schemes are incompatible with exotic monomers that diverge structurally from canonical α-amino acids and are suboptimal substrates for the ribosome. To enable the ribosomal incorporation of such exotic monomers, a two-step solution is needed: A) Engineering an aaRS to acylate its cognate tRNA with the exotic monomer, without relying on ribosomal translation as a readout, and B) Subsequent engineering of the ribosome to accept the resulting acylated tRNA for translation. Here, we report a platform for aaRS engineering that directly selects for tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the associated barcode-containing tRNAs from an oxidative treatment designed to damage the 3′-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of theM. alvuspyrrolysyl-tRNA synthetase from a naïve library that charge noncanonical amino acids.
Exploring the impact of microbial manipulation on the early development of kelp (Saccharina latissima) using an ecological core microbiome framework
Authors: Park Jungsoo , Schenk Siobhan , Davis Katherine , Clark Jennifer , Parfrey Laura Wegener
Published: December 2023

Kelp cultivation is a rapidly expanding economic sector, as kelp are valued for a wide range of commercial products and for restoration of kelp forest ecosystems. Microbes associated with kelp and other macroalgae play a critical role in processes such as nutrient exchange, chemical signaling, and defense against pathogens. Thus, manipulating the microbiome to enhance macroalgal growth and resilience is a promising, but largely untested, tool in sustainable kelp cultivation. The core microbiome hypothesis suggests that bacteria that are consistently found on a host (the core microbes) are likely to have a disproportionate impact on host biology, making them an attractive target for microbiome manipulation. Here, we surveyed wildSaccharina latissimaand their surrounding environment to identify core bacterial taxa, compared to cultivated kelp, and experimentally tested the effect of cultured bacterial isolates on kelp development. We find that core bacteria are nearly absent in cultivated juvenile sporophytes in nurseries but eventually colonized after outplanting kelp to ocean farm sites. We find that bacterial inoculants can have both positive and negative effects on kelp development. In line with predictions from the core microbiome hypothesis, we find a positive correlation between the frequency of the bacterial genus in the wild and the bacterial effect on the number of sporophytes in kelp co-culture experiments.

IMPORTANCE

The core microbiome hypothesis suggests that symbiotic microorganisms consistently associated with hosts have functional effects on host biology and health. However, there is a lack of evidence to either support or refute this idea. This study surveys the distribution of bacteria on wild and cultivated kelp to identify the core microbiome and tests the ability of bacterial isolates cultured from the surface of wild kelp to influence kelp growth and development in laboratory microbial manipulation experiments. The frequency of bacterial genera on wild kelp was positively correlated with influence on kelp development in laboratory experiments, providing support for the core microbiome hypothesis.
Activity of Antibiotics againstBurkholderia cepaciacomplex in Artificial Sputum Medium
Authors: Shukla Anusha , Rodriguez Shade , Brennan-Krohn Thea
Published: December 2023

Burkholderia cepaciacomplex (Bcc) is a collection of intrinsically drug-resistant gram-negative bacteria that cause life-threatening pulmonary disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in people with Bcc infections, likely due in part to the significant differences betweenin vitrotesting conditions and the environment in which Bcc grow in the lungs of people with CF. We tested the activity of six commonly used antibiotics against two clinical Bcc strains grown to high density in an artificial sputum medium in order to assess their activity in conditions mimicking those foundin vivo. There were major discrepancies between standard susceptibility results and activity in our model, with some antibiotics, including ceftazidime, showing minimal activity despite low MICs, while others, notably tobramycin, were more active in high-density growth conditions than in standard assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.
The HmrABCX pathway regulates the transition between motile and sessile lifestyles inCaulobacter crescentusby a HfiA-independent mechanism
Authors: Zappa Sébastien , Berne Cecile , Morton Robert I. , De Stercke Jonathan , Brun Yves V.
Published: December 2023

Through its cell cycle, the bacteriumCaulobacter crescentusswitches from a motile, free-living state, to a sessile surface-attached cell. During this coordinated process, cells undergo irreversible morphological changes, such as shedding of their polar flagellum and synthesis of an adhesive holdfast at the same pole. In this work, we used genetic screens to identify genes involved in the regulation of the motile to sessile lifestyle transition. We identified a predicted hybrid histidine kinase that inhibits biofilm formation and activates the motile lifestyle: HmrA (Holdfast andmotility regulator A). Genetic screens and genomic localization led to the identification of additional genes that regulate the proportion of cells harboring an active flagellum or a holdfast and that form a putative phosphorelay pathway with HmrA. Further genetic analysis indicates that the Hmr pathway is independent of the holdfast synthesis regulator HfiA and may impact c-di-GMP synthesis through the diguanylate cyclase DgcB pathway. Finally, we provide evidence that the Hmr pathway is involved in the regulation of sessile-to-motile lifestyle as a function of environmental stresses, namely excess copper and non-optimal temperatures.

IMPORTANCE

Complex communities attached to a surface, or biofilms, represent the major lifestyle of bacteria in the environment. Such a sessile state enables its inhabitants to be more resistant to adverse environmental conditions. Thus, having a deeper understanding of the underlying mechanisms that regulate the transition between the motile and the sessile states could help design strategies to improve biofilms when they are beneficial or impede them when they are detrimental. ForC. crescentusmotile cells, the transition to the sessile lifestyle is irreversible, and this decision is regulated at several levels. In this work, we describe a putative phosphorelay that promotes the motile lifestyle and inhibits biofilm formation, providing new insights into the control of adhesin production that leads to the formation of biofilms.