bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic regulation of cell-type specific chromatin accessibility shapes the etiology of brain diseases
Authors: Zeng Biao , Bendl Jaroslav , Deng Chengyu , Lee Donghoon , Misir Ruth , Reach Sarah M. , Kleopoulos Steven P. , Auluck Pavan , Marenco Stefano , Lewis David A. , Haroutunian Vahram , Ahituv Nadav , Fullard John F. , Hoffman Gabriel E. , Roussos Panos
Published: March 2023

Nucleotide variants in cell type-specific gene regulatory elements in the human brain are major risk factors of human disease. We measured chromatin accessibility in sorted neurons and glia from 1,932 samples of human postmortem brain and identified 34,539 open chromatin regions with chromatin accessibility quantitative trait loci (caQTL). Only 10.4% of caQTL are shared between neurons and glia, supporting the cell type specificity of genetic regulation of the brain regulome. Incorporating allele specific chromatin accessibility improves statistical fine-mapping and refines molecular mechanisms underlying disease risk. Using massively parallel reporter assays in induced excitatory neurons, we screened 19,893 brain QTLs, identifying the functional impact of 476 regulatory variants. Combined, this comprehensive resource captures variation in the human brain regulome and provides novel insights into brain disease etiology.

One sentence summary

Cell-type specific chromatin accessibility QTL reveals regulatory mechanisms underlying brain diseases.
Disentangling the effects of Corticotrophin Releasing Factor and GABA release from the ventral bed nucleus of the stria terminalis on ethanol self-administration in mice
Authors: Gianessi C.A. , Gereau G.B. , Haun H.L. , Pati D. , Sides T. , D’Ambrosio S.L. , Boyt K. , Kelson W.P. , Hodge C.W. , Kash T.L.
Published: March 2023

Excessive alcohol use causes a great deal of harm and negative health outcomes. Corticotrophin releasing factor (CRF), a stress-related neuropeptide, has been implicated in binge ethanol intake and ethanol dependence. CRF containing neurons in the bed nucleus of the stria terminalis (BNSTCRF) can control ethanol consumption. These BNSTCRFneurons also release GABA, raising the question, is it CRF or GABA release or both that is controlling alcohol consumption. Here, we used viral vectors to separate the effects of CRF and GABA release from BNSTCRFneurons on the escalation of ethanol intake in an operant self-administration paradigm in male and female mice. We found that CRF deletion in BNST neurons reduces ethanol intake in both sexes, with a stronger effect in males. For sucrose self-administration there was no effect of CRF deletion. Suppression of GABA release, via knockdown of vGAT, from BNSTCRFproduced a transient increase in ethanol operant self-administration following in male mice, and reduced in motivation to work for sucrose on a progressive ratio schedule of reinforcement in a sex-dependent manner. Together, these results highlight how different signaling molecules from the same populations of neurons can bidirectionally control behavior. Moreover, they suggest that BNST CRF release is important for high intensity ethanol drinking that precedes dependence, whereas GABA release from these neurons may play a role in regulating motivation.
Iodidimonas, a bacterium unable to degrade hydrocarbons, thrives in a bioreactor treating oil and gas produced water
Authors: Acharya Shwetha M. , Enalls Brandon C. , Walian Peter J , Van Houghton Brett D. , Rosenblum James S. , Cath Tzahi Y. , Tringe Susannah G. , Chakraborty Romy
Published: March 2023

Iodidimonasis a genus recently described in bioreactors treating oil and gas produced water and in iodide rich brines. Besides the ability to oxidize iodine, little is known about the metabolic capabilities that enableIodidimonassp. to occupy this unique ecological niche. We isolated, characterized, and sequenced three strains belonging to theIodidimonasgenus from the sludge of a membrane bioreactor treating produced water. We describe the genomic features of these isolates and compare them with the only other four isolate genomes reported from this genus, as well as a metagenome-assembled genome from the source bioreactor. To survive in the produced water,Iodidimonasisolates had several genes associated with mitigating salinity, heavy metal and organic compound stress. While the isolates could utilize a wide variety of carbon substrates, they failed to degrade aliphatic or aromatic hydrocarbons, consistent with the lack of genes associated with common hydrocarbon degradation pathways in their genomes. We hypothesize these microbes may lead a scavenging lifestyle in the bioreactor and similar iodide-rich brines.

Importance

Occupying a niche habitat and having few representative isolates, genusIodidimonasis a relatively understudied Alphaproteobacterial group. This genus has garnered attention due to its ability to corrode pipes in iodine production facilities and generate iodinated organic compounds during treatment of oil and gas produced water. The iodinated organic compounds are likely to be carcinogenic and may pose issues with recycling the treated water. Hence, detailed characterization of the metabolic potential of these isolates is not only of economic importance, but also sheds light on adaptation of this microbe to its environmental niche.
Mutational signatures in wild typeEscherichia colistrains reveal dominance of DNA polymerase errors
Authors: Garushyants Sofya K. , Sane Mrudula , Selifanova Maria V. , Agashe Deepa , Bazykin Georgii A. , Gelfand Mikhail S.
Published: March 2023

While mutational processes operating in theEscherichia coligenome have been revealed by multiple laboratory experiments, the contribution of these processes to accumulation of bacterial polymorphism and evolution in natural environments is unknown. To address this question, we reconstruct signatures of distinct mutational processes from experimental data onE. colihypermutators, and ask how these processes contribute to differences between naturally occurringE. colistrains. We show that both mutations accumulated in the course of evolution of wild type strains in nature and in the lab-grown non-mutator laboratory strains are explained predominantly by the low fidelity of DNA polymerases II and III. By contrast, contributions specific to disruption of DNA repair systems cannot be detected, suggesting that temporary accelerations of mutagenesis associated with such disruptions are unimportant for within-species evolution. These observations demonstrate that accumulation of diversity in bacterial strains in nature is predominantly associated with errors of DNA polymerases.
Live-cell imaging uncovers the relationship between histone acetylation, transcription initiation, and nucleosome mobility
Authors: Saxton Matthew N. , Morisaki Tatsuya , Krapf Diego , Kimura Hiroshi , Stasevich Timothy J.
Published: March 2023

Post-translational protein modifications play an important role in the regulation of gene dynamics. Certain modifications, such as histone acetylation and RNA polymerase II phosphorylation, are associated with transcriptionally active chromatin. However, the spatial and temporal relationship between chromatin and post-translational protein modifications, and how these dynamics facilitate selective gene expression, remain poorly understood. In this study, we address this problem by developing a general methodology for quantifying in live cells the dynamics of chromatin across multiple time and length scales in the context of residue-specific protein modifications. By combining Fab-based labeling of endogenous protein modifications with single-molecule imaging, we track the dynamics of chromatin enriched with histone H3 Lysine-27 acetylation (H3K27ac) and RNA polymerase II Serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals chromatin enriched with H3K27ac is separated from chromatin enriched with RNAP2-Ser5ph. Furthermore, in these separated sites, we show the presence of the two modifications are inversely correlated with one another on the minutes timescale. We then track single nucleosomes in both types of sites on the sub-second timescale and again find evidence for distinct and opposing changes in their diffusive behavior. While nucleosomes diffuse ∼15% faster in chromatin enriched with H3K27ac, they diffuse ∼15% slower in chromatin enriched with RNAP2-Ser5ph. Taken together, these results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each modification marks distinct sites that are transcriptionally poised or active, respectively.
Identifying promoter sequence architectures via a chunking-based algorithm using non-negative matrix factorisation
Authors: Nikumbh Sarvesh , Lenhard Boris
Published: March 2023

Core promoters are stretches of DNA at the beginning of genes that contain information that facilitates the binding of transcription initiation complex. Different functional subsets of genes have core promoters with distinct architectures and characteristic motifs. Some of these motifs inform the selection of transcription start sites (TSS). By discovering motifs with fixed distances from known TSS positions, we could in principle classify promoters into different functional groups.

Due to the variability and overlap of architectures, promoter classification is a difficult task that requires new approaches. In this study, we present a new method based on non-negative matrix factorisation (NMF) and the associated software called seqArchR that clusters promoter sequences based on their motifs at near-fixed distances from a reference point, such as TSS. When combined with experimental data from CAGE, seqArchR can efficiently identify TSS-directing motifs, including known ones like TATA, DPE, and nucleosome positioning signal, as well as novel lineage-specific motifs and the function of genes associated with them. By using seqArchR on developmental time courses, we reveal how relative use of promoter architectures changes over time with stage-specific expression.

seqArchR is a powerful tool for initial genome-wide classification and functional characterization of promoters. Its use cases are more general: it can also be used to discover any motifs at near-fixed distances from a reference point, even if they are present in only a small subset of sequences. seqArchR is available athttp://www.bioconductor.org/packages/seqArchR.
Probing inter-areal computations with a cellular resolution two-photon holographic mesoscope
Authors: Abdeladim Lamiae , Shin Hyeyoung , Jagadisan Uday K. , Ogando Mora B. , Adesnik Hillel
Published: March 2023

Brain computation depends on intricately connected yet highly distributed neural networks. Due to the absence of the requisite technologies, causally testing fundamental hypotheses on the nature of inter-areal processing have remained largely out-of-each. Here we developed the first two photon holographic mesoscope, a system capable of simultaneously reading and writing neural activity patterns with single cell resolution across large regions of the brain. We demonstrate the precise photo-activation of spatial and temporal sequences of neurons in one brain area while reading out the downstream effect in several other regions. Investigators can use this new platform to understand feed-forward and feed-back processing in distributed neural circuits with single cell precision for the first time.
Quantitative Nanohistology of aging dermal collagen
Authors: Huang Sophia , Strange Adam , Maeva Anna , Siddiqui Samera , Bastien Phillipe , Aguayo Sebastian , Vaez Mina , Montagu-Pollock Hubert , Ghibaudo Marion , Potter Anne , Pageon Herve , Bozec Laurent
Published: March 2023

While the external signs of skin aging have been well-defined throughout history, much less is known about aging within the ultrastructure of our skin. Our skin, the largest organ in our body, is structured by collagen through fibrils or large sheets. With the increased use of nanometrology tools in histology, it is now possible to explore how the aging process affects collagen at its most fundamental level, the collagen fibril. Here, we show how atomic force microscopy-based quantitative nanohistology can differentiate skin from different age groups and anatomical sites. Following the definition of specific collagen biomarkers at the nanoscale, we used a segmentation approach to quantify the prevalence of 4 structural biomarkers over a dataset of 42,000 images (30 donors) complemented by extensive nanomechanical analyses (30,000 indentation curves) on histological sections. Our results demonstrate that specific age-related collagen fingerprints could be found when comparing the % prevalence of each marker between the papillary and reticular dermis. A case of abnormal biological aging validated our markers and nanohistology approach. This first extensive study focusing on defining signs of dermal aging at the nanoscale proves we are all unique to our dermal collagen ultrastructure.
Capacitance measurements for assessing DNA origami nanostructures
Authors: Walawalkar Vismaya , Sajal Md. Sakibur , Gilpin Yann , Dandin Marc , Taylor Rebecca E.
Published: March 2023

Nanostructures fabricated with DNA are emerging as a practical approach for applications ranging from advanced manufacturing to therapeutics. To support the strides made in improving accessibility and facilitating commercialization of DNA nanostructure applications, we identify the need for a rapid characterization approach that aids nanostructure production. In our work, we introduce a low-fidelity characterization approach that provides an interdependent assessment of DNA origami formation, concentration and morphology using capacitance sensing. Change in charge is one of the transduction methods to determine capacitive loading on a substrate. It is known that cations in the solution stabilize DNA origami nanostructures. So, we hypothesized that the presence of cations and nanostructures in a buffer solution can induce capacitance change that is distinctive of the nanostructure present. In this study we were able to detect a change in the capacitance when the nanostructure solution was deposited on our capacitance sensor, and we could distinguish between pre-annealed and annealed structures at concentrations less than 15 nM. The capacitance measurements were affected by the concentration of Mg2+ions in the solution, the staple-to-scaffold stoichiometric ratio of the nanostructure and the nanostructure morphology. Maintaining a 12.5 mM Mg2+concentration in the nanostructure buffer, we discover a linear relationship between the relative capacitance change and the nanostructure concentration from 5 nM to 20 nM, which we call the characteristic curve. We find distinct characteristic curves for our three nanostructures with distinct morphologies but similar molecular weight - a rectangular plate, a sphere and a rod. Given that we can distinguish nanostructure formation, concentration and morphology, we expect that capacitance measurement will emerge as an affordable and rapid approach for quality control for nanostructure production.
Incremental improvements in tractometry-based brain-age modeling with deep learning
Authors: Rokem Ariel , Qiao Joanna , Yeatman Jason D. , Richie-Halford Adam
Published: March 2023

Multivariate measurements of human brain white matter (WM) with diffusion MRI (dMRI) provide information about the role of WM in a variety of cognitive functions and in brain health. Statistical models take advantage of the regularities in these data to make inferences about individual differences. For example, dMRI data provide the basis for accurate brain-age models – models that predict the chronological age of participants from WM tissue properties. Deep learning (DL) models are powerful machine learning models, which have been shown to provide benefits in many multivariate analysis settings. We investigated whether DL would provide substantial improvements for brain-age models based on dMRI measurements of WM in a large sample of children and adolescents. We found that some DL models fit the data better than a linear baseline, but the differences are small. In particular, recurrent neural network architectures provide up to ∼6% improvement in accuracy. This suggests that information about WM development is mostly accessible with linear models, and does not require the additional invariance and non-linearity offered by DL models. However, in some applications this incremental improvement may prove critical. We provide open-source software that fits DL models to dMRI data (https://yeatmanlab.github.io/AFQ-Insight).
Mosquito thermal tolerance is remarkably constrained across a large climatic range
Authors: Couper Lisa I. , Farner Johannah E. , Lyberger Kelsey P. , Lee Alexandra S. , Mordecai Erin A.
Published: March 2023

How mosquitoes may respond to rapid climate warming remains unknown for most species, but will have major consequences for their future distributions, with cascading impacts on human well-being, biodiversity, and ecosystem function. We investigated the adaptive potential of a wide-ranging mosquito species,Aedes sierrensis, across a large climatic gradient by conducting a common garden experiment measuring the thermal limits of mosquito life history traits. Although field-collected populations originated from vastly different thermal environments that spanned over 1,200 km, we found remarkably limited variation in upper thermal tolerance between populations, with the upper thermal limits of fitness varying by <1°C across the species range. For one life history trait—pupal development rate—we did detect significant variation in upper thermal limits between populations, and this variation was strongly correlated with source temperatures, providing evidence of local thermal adaptation for pupal development. However, we found environmental temperatures already regularly exceed our highest estimated upper thermal limits throughout most of the species range, suggesting limited potential for mosquito thermal tolerance to evolve on pace with warming. Strategies for avoiding high temperatures such as diapause, phenological shifts, and behavioral thermoregulation are likely important for mosquito persistence.
Feature selection followed by a novel residuals-based normalization simplifies and improves single-cell gene expression analysis
Authors: Singh Amartya , Khiabanian Hossein
Published: March 2023

Normalization is a crucial step in the analysis of single-cell RNA-sequencing (scRNA-seq) counts data. Its principal objectives are to reduce the systematic biases primarily introduced through technical sources and to transform the data to make it more amenable for application of established statistical frameworks. In the standard workflows, normalization is followed by feature selection to identify highly variable genes (HVGs) that capture most of the biologically meaningful variation across the cells. Here, we make the case for a revised workflow by proposing a simple feature selection method and showing that we can perform feature selection before normalization by relying on observed counts. We highlight that the feature selection step can be used to not only select HVGs but to also identify stable genes. We further propose a novel variance stabilization transformation inclusive residuals-based normalization method that in fact relies on the stable genes to inform the reduction of systematic biases. We demonstrate significant improvements in downstream clustering analyses through the application of our proposed methods on biological truth-known as well as simulated counts datasets. We have implemented this novel workflow for analyzing high-throughput scRNA-seq data in an R package called Piccolo.
Emergence of rhythmic chunking in complex stepping of mice
Authors: Hirokane Kojiro , Nakamura Toru , Kubota Yasuo , Hu Dan , Yagi Takeshi , Graybiel Ann M. , Kitsukawa Takashi
Published: March 2023

Motor chunking is important for motor execution, allowing atomization and efficiency of movement sequences. However, it remains unclear why and how chunks contribute to motor execution. To analyze the structure of naturally occurring chunks, we trained mice to run in a complex series of steps and identified the formation of chunks. We found that intervals (cycle) and the positional relationship between the left and right limbs (phase) of steps inside the chunks, unlike those outside the chunks, were consistent across occurrences. Further, licking by the mice was also more periodic and linked to the specific phases of limb movements within the chunk. Based on these findings, we propose therhythm chunking hypothesis, whereby within chunks, the repetitive movements of many body parts are linked by the rhythm parameters: cycle and phase. The computational complexity of movement may thereby be reduced by adjusting movements as the combination of rhythms.
A high sugar diet, but not obesity, reduces female fertility inDrosophila melanogaster
Authors: Nunes Rodrigo Dutra , Drummond-Barbosa Daniela
Published: March 2023

Many studies fromDrosophilato humans show a strong link between obesity and reduced fertility. However, obesity is often induced by changes in diet or eating behavior, such that it remains unclear whether reduced fertility is a consequence of obesity itself, diet, or both. Here, we report that a high sugar diet reducesDrosophilafemale fertility by increasing death of early germline cysts (prior to follicle formation) and degeneration of vitellogenic follicles; that obesity in and of itself does not impair fertility; and that high glucose levels closely correlate with reduced fertility on a high sugar diet. Females on a high sugar diet rapidly develop obesity (and display high glycogen, glucose, and trehalose levels, and insulin resistance) and decreased fertility. In stark contrast to high-sugar-obese females, females in which similar levels of obesity are induced by adipocyte-specific knockdown of anti-obesity genesbrummeroradiposehave normal fertility and sugar metabolic indicators. Remarkably, females on a high sugar diet supplemented with a separate source of water also have normal fertility and glucose levels, despite persistent obesity, high glycogen and trehalose levels, and insulin resistance markers. These results strengthen our conclusion that obesity itself does not impair fertility, show an inverse correlation between high glucose levels and fertility, and demonstrate that insulin signaling levels remain sufficiently high to maintain insulin-dependent processes during oogenesis irrespective of insulin resistance markers.
Enrichment and delivery of target proteins into the cell cytosolviaOuter Membrane Vesicles
Authors: Wan Huan , Tao Zhiqing , Zhao XiaoLing , Wang Guan , Chen Yihao , Zhang Juan , Zhang Xu , Liu Maili , Jiang Guosheng , He Lichun
Published: March 2023

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods, have drawbacks such as the requirement of high concentrationin vitroprepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/ml) of several target proteins into outer membrane vesicles (OMVs) ofE. colito employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency arranging from 90-97% for different cell lines. Moreover, the free penetration of molecules less than 600 Dalton across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.
Polygenic outcomes of sexually antagonistic selection
Authors: Muralidhar Pavitra , Coop Graham
Published: March 2023

Sexual antagonism occurs when males and females have different fitness optima for a phenotype, but are constrained from evolving to these optima because of their shared genome. We study the response of a polygenic phenotype to the onset of sexually antagonistic selection, modeling a phenotype initially under stabilizing selection around an optimum, followed by a sudden divergence of the male and female optima. We observe rapid phenotypic evolution to these new optima via small changes in allele frequencies genome-wide. We study the role of sex chromosomes in this divergence and find that, in the absence of dosage compensation, the X chromosome favors evolution toward the female optimum, inducing co-evolutionary male-biased responses on the autosomes. However, dosage compensation obscures the female-biased interests of the X, causing it to contribute equally to male and female phenotypic change. In both scenarios, we see little effect of dominance in the genetic variation utilized by the X chromosome vs. the autosomes. We go on to examine the dynamics of stabilizing selection once the male and female optima have been reached, exploring a subtle mechanism through which the X chromosome, via the Bulmer effect, can cause higher equilibrium phenotypic variance in males than females. Finally, we consider how sexual antagonistic selection might persist across longer time scales, demonstrating that random fluctuations in an adaptive landscape can generate prolonged intragenomic conflict. Overall, our results provide insight into the response of complex phenotypes to sexually antagonistic selection and the evolution of sexual dimorphism.
Biliatresone treatment of pregnant mice causes changes in bile metabolism and liver inflammation in their offspring
Authors: Gupta Kapish , Xu Jimmy P. , Diamond Tamir , De Jong Iris , Glass Andrew , Llewellyn Jessica , Theise Neil D. , Winkler Jeffrey D. , Behrens Edward M. , Mesaros Clementina , Wells Rebecca G.
Published: March 2023

Background & Aims

Biliary atresia is a neonatal disease characterized by bile duct and liver damage, fibrosis, inflammation and abnormal bile metabolism. It appears to result from a prenatal exposure that spares the mother and affects the fetus. Our aim was to define the phenotype in neonatal mice after maternal exposure to low-dose biliatresone, a plant toxin implicated in biliary atresia in livestock.

Methods

Pregnant mice were treated orally with low-doses of biliatresone. Histological changes, bile acid profiles and immune profiles were analyzed in postnatal day 5 and 21 pups born to treated mothers.

Results

The pups of mothers treated with this dose of biliatresone had no evidence of significant liver or ductular injury or fibrosis at postnatal day 5 or 21 and they grew normally. However, serum levels of glycocholic acid were elevated at postnatal day 5, suggesting altered bile metabolism, and bile metabolism became increasingly abnormal through postnatal day 21, with enhanced glycine conjugation of bile acids. There was also immune cell activation observed in the liver at postnatal day 21.

Conclusion

Prenatal exposure to low doses of an environmental toxin can cause liver inflammation and aberrant bile metabolism even in the absence of histological changes.

Lay summary

Prenatal exposure to low doses of an environmental toxin can cause changes in bile metabolism in neonatal mice.
Epigenomic divergence underlies sequence polymorphism and the evolutionary fate of duplicate paralogs inA. thaliana
Authors: Kenchanmane Raju Sunil K. , Lensink Mariele , Kliebenstein Daniel J. , Niederhuth Chad , Monroe Grey
Published: March 2023

Processes affecting rates of sequence polymorphism are fundamental to molecular evolution and the evolutionary fate of gene duplicates. The relationship between gene activity and sequence polymorphism can influence the likelihood that functionally redundant gene copies are co-maintained in stable evolutionary equilibria versus other outcomes such as neo-functionalization. Here we investigate genic variation in epigenome-associated polymorphism rates inArabidopsis thalianaand consider whether these affect the evolution of gene duplicates. We compared the frequency of sequence polymorphism and patterns of genetic differentiation between genes classified by exon methylation patterns: unmethylated (unM), gene-body methylated (gbM), and transposon-like methylated (teM) states, which reflect divergence in gene expression. We found that the frequency of polymorphism was higher in teM (transcriptionally repressed, tissue-specific) genes and lower in gbM (active, constitutively expressed) genes. Comparisons of gene duplicates were largely consistent with genome-wide patterns - gene copies that exhibit teM tend to accumulate higher sequence polymorphism, evolve faster, and are in chromatin states associated with reduced DNA repair. This relationship between expression, the epigenome, and polymorphism may lead to the breakdown of equilibrium states that would otherwise maintain genetic redundancies. Epigenome-mediated polymorphism rate variation may therefore aid the pseudogenization of duplicate paralogs or increase the evolution of novel gene functions in duplicate paralogs maintained over evolutionary time.
Respiration is essential for aerobic growth ofZymomonas mobilisZM4
Authors: Felczak Magdalena M. , TerAvest Michaela A.
Published: March 2023

Zymomonas mobilisis an alpha-proteobacterium that is a promising platform for industrial scale production of biofuels or valuable products due to its efficient ethanol fermentation and low biomass generation.Z. mobilishas also intriguing physiology, sometimes difficult to explain by the rules and strategies commonly observed in other bacteria. One of the most mysterious features ofZ. mobilisis its growth in oxic conditions.Z. mobilisis an aerotolerant bacterium that encodes a complete respiratory electron transport chain but the benefit of respiration for growth in oxic conditions has never been confirmed, despite decades of research. Quite the opposite, growth and ethanol production of WTZ. mobilisis poor in oxic conditions indicating that it does not benefit from oxidative phosphorylation. Additionally, in previous studies, aerobic growth improved significantly when respiratory genes were disrupted (ndh) or acquired point mutations (cydA, cydB)even if respiration was significantly reduced by these changes. Here, we obtained clean deletions of respiratory genesndhandcydAB, individually and in combination, and showed, for the first time, that deletion ofcydABcompletely inhibited O2respiration and dramatically reduced growth in oxic conditions. Both respiration and aerobic growth were restored by expressing a heterologous, water-forming NADH oxidase,noxE. This result shows that the main role of the electron transport chain inZ. mobilisis reducing the toxicity of molecular oxygen, helping to explain why it is beneficial forZ. mobilisto use electron transport chain complexes that contribute little to oxidative phosphorylation.

Importance

A key to producing next generation biofuels is to engineer microbes that efficiently convert non-food materials into drop-in fuels and to engineer microbes effectively we must understand their metabolism thoroughly.Zymomonas mobilisis a bacterium that is a promising candidate biofuel producer but its metabolism remains poorly understood, especially its metabolism when exposed to oxygen. AlthoughZ. mobilisrespires with oxygen, its aerobic growth is poor and disruption of genes related to respiration counterintuitively improves aerobic growth. This unusual result has sparked decades of research and debate regarding the function of respiration inZ. mobilis. Here, we used a new set of mutants to determine that respiration is essential for aerobic growth and likely protects the cells from oxidative damage caused by molecular oxygen. These results indicate that respiration has a non-canonical function inZ. mobilisand expand our understanding of the role of respiration in metabolism and oxidative stress.
Inhibition of hostN-myristoylation compromises the infectivity of SARS-CoV-2 due to Golgi-bypassing egress from lysosomes and endoplasmic reticulum
Authors: Saber Saber H. , Shaker Mohammed R. , Sng Julian , Yak Nyakuoy , Morrison Sean D. , Pars Selin , Zheng Huiwen , Pietrogrande Giovanni , Binder Tobias , Lu Siyuan , Floetenmeyer Matthias , Ojha Ravi , Quirin Tania , Ruuska Janika , Smura Teemu , Strandin Tomas , Kant Ravi , Kareinen Lauri , Sironen Tarja , Talbo Gert Hoy , Zhu Yanshan , Short Kirsty R. , Mar Jessica , Kallemeijn Wouter W. , Tate Edward W. , Solari Roberto , van Waardenberg Ashley J. , Vapalahti Olli , Wolvetang Ernst , Balistreri Giuseppe , Joensuu Merja
Published: March 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, remains a global health concern despite vaccines, neutralizing antibodies, and antiviral drugs. Emerging mutations can reduce the effectiveness of these treatments, suggesting that targeting host cell factors may be a valuable alternative.N-myristoyltransferases (NMT) are essential enzymes for proteinN-myristoylation, affecting stability, interaction, localization, and function of numerous proteins. We demonstrate that selective inhibition of host cell NMT decreases SARS-CoV-2 infection by 90% in human lung and primary nasal epithelial cells, and choroid plexus-cortical neuron organoids. NMT inhibition does not affect viral entry, replication or release, but impairs the maturation and incorporation of viral envelope proteins into newly assembled virions, leading to compromised infectivity of released virions. The inhibition of host NMT triggers a Golgi-bypassing pathway for SARS-CoV-2 progeny virion egress, which occurs through endoplasmic reticulum and lysosomal intermediates.
Underlying causes for prevalent false positives and false negatives in STARR-seq data
Authors: Ni Pengyu , Wu Siwen , Su Zhengchang
Published: March 2023

STARR-seq and its variants have been widely used to characterize enhancers. However, it has been reported that up to 87% of STARR peaks are located in repressive chromatins and are not functional in the tested cells. While some of the STARR peaks in repressive chromatins might be active in other cell/tissue types, some others might be false positives. Meanwhile, many active enhancers may not be identified by the current STARR-seq methods. However, the prevalence of and underlying causes for the artifacts are not fully understood. Based on predictedcis-regulatory modules (CRMs) and non-CRMs in the human genome as well as predicted active CRMs and non-active CRMs in a few human cell lines with STARR-seq data available, we reveal prevalent false positives and false negatives in STARR peaks and possible underlying causes. Our results will help design strategies to improve STARR-seq methods and interpret the results.
Chromatin Accessibility and Pioneer Factor FOXA1 Shape Glucocorticoid Receptor Action in Prostate Cancer
Authors: Helminen Laura , Huttunen Jasmin , Aaltonen Niina , Niskanen Einari A. , Palvimo Jorma J. , Paakinaho Ville
Published: March 2023

Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of theNR3C1(gene encoding GR) expressionviathe corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300’s enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.
Differential expression and function of SVIP in breast cancer cell lines andin silicoanalysis of its expression and prognostic potential in human breast cancer
Authors: Atalay Şahar Esra , Ballar Kirmizibayrak Petek
Published: March 2023

The heterogeneity of cancer strongly suggests the need to explore additional pathways to target. As cancer cells have increased proteotoxic stress, targeting proteotoxic stress-related pathways such as endoplasmic reticulum stress is attracting attention as new anticancer treatments. One of the downstream responses to endoplasmic reticulum stress is endoplasmic reticulum-associated degradation (ERAD), a major degradation pathway that facilitates proteasome-dependent degradation of unfolded or misfolded proteins. Recently, SVIP, an endogenous ERAD inhibitor, has been implicated in cancer progression, especially in glioma, prostate, and head and neck cancers. Here, the data of several RNA-seq and gene array studies were combined to evaluate the SVIP gene expression analysis on a variety of cancers, with a particular focus on breast cancer. SVIP was found to be overexpressed in primary breast tumors compared to normal tissues correlated well with its promoter methylation status and genetic alterations. Similarly, immunoblotting analysis showed that SVIP was expressed significantly higher in breast cancer cell lines compared to non-tumorigenic epithelial cell line. On the other hand, the expression of the key proteins of gp78-mediated ERAD did not exhibit such a pattern. Interestingly, silencing of SVIP enhanced the proliferation of p53 wt MCF7 cells but not p53 mutant T47D cells, however increased migration ability of both cell lines. Interestingly, SVIP expression is high in primary breast tumors but low in breast metastatic tumors. This correlates well with a lower probability of survival of breast cancer patients with lower SVIP expression compared to the patients with overexpressed SVIP. Overall, our data revealing the differential expression and function of SVIP on breast cancer cell lines together within silicodata analysis suggest that SVIP may have complex functions in breast cancer progression and has the potential to be a therapeutic target for breast cancer.
Phenotypic link between protein efficiency and pig welfare suggests no apparent trade-offs for mitigating nitrogen pollution
Authors: Roch Lea , Ewaoluwagbemiga Esther Oluwada , Kasper Claudia
Published: March 2023

Pig manure contributes significantly to environmental pollution through nitrogen compounds. Reducing protein in feed can help, but it may lead to damaging behaviors if pigs’ nutritional needs are not met. Breeding pigs for higher protein efficiency (PE) is a long-term solution to reduce nitrogen pollution, but concerns about pig welfare remain. We studied 95 pigs involved in a project on the genetic basis of PE on a 20% protein restricted diet to investigate the phenotypic connection between PE and welfare. These pigs represented natural PE variations in the population. At around 100 days, before their PE was known, we observed their behaviors. Only three pigs engaged in tail biting and manipulation of vulnerable regions, but this was not associated with PE. There was no clear link between PE and manipulating pen mates’ less vulnerable regions. Such behaviors are normal but can cause stress and injury if carried out excessively due to boredom or stress. Overall, pigs with higher PE showed no major behavioral abnormalities in this study. Considering the lack of genetic knowledge, the risk of increased harmful behaviors when selecting for higher PE appears low when inferred from this purely phenotypic association.
Widespread CRISPR repeat-like RNA regulatory elements in CRISPR-Cas systems
Authors: Shmakov Sergey A. , Barth Zachary K. , Makarova Kira S. , Wolf Yuri I. , Brover Vyacheslav , Peters Joseph E. , Koonin Eugene V.
Published: March 2023

CRISPR-casloci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR-casloci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-casloci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions ofcasgenes, in particularcas8, or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.
Insights into the function of the chloroplastic ribosome-associated GTPase HflX inArabidopsis thaliana
Authors: Mehrez Marwa , Lecampion Cécile , Ke Hang , Gorsane Faten , Field Ben
Published: March 2023

Ribosome-associated GTPases are conserved enzymes that participate in ribosome biogenesis and ribosome function. In bacteria, recent studies have identified HflX as a ribosome-associated GTPase that is involved in both ribosome biogenesis and recycling under stress conditions. Plants possess a chloroplastic HflX homolog, but its function remains unknown. Here, we characterised the role of HflX in the plantArabidopsis thaliana. Our findings demonstrate that HflX does not have a detectable role in plant growth and development, nor does it play a distinct role in acclimation to several different stresses, including heat, manganese, cold, and salt stress. However, we found that HflX is required for plant resistance to chloroplast translational stress mediated by the antibiotic lincomycin. Our results suggest that HflX is a chloroplast ribosome-associated protein that may play a role in the surveillance of translation. These findings provide new insight into the function of HflX as a ribosome-associated GTPase in plants and highlight the importance of investigating conserved proteins in different organisms to gain a comprehensive understanding of their biological roles.
Training Doctoral Students in Critical Thinking and Experimental Design using Problem-based Learning
Authors: Schaller Michael D. , Gencheva Marieta , Gunther Michael R. , Weed Scott A.
Published: March 2023

Background

Traditionally, doctoral student education in the biomedical sciences relies on didactic coursework to build a foundation of scientific knowledge and an apprenticeship model of training in the laboratory of an established investigator. Recent recommendations for revision of graduate training include the utilization of graduate student competencies to assess progress and the introduction of novel curricula focused on development of skills, rather than accumulation of facts. Evidence demonstrates that active learning approaches are effective. Several facets of active learning are components of problem-based learning (PBL), which is a teaching modality where student learning is self-directed toward solving problems in a relevant context. These concepts were combined and incorporated in creating a new introductory graduate course designed to develop scientific skills (student competencies) in matriculating doctoral students using a PBL format.

Methods

Evaluation of course effectiveness was measured using the principals of the Kirkpatrick Four Level Model of Evaluation. At the end of each course offering, students completed evaluation surveys on the course and instructors to assess their perceptions of training effectiveness. Pre- and post-tests assessing students’ proficiency in experimental design were used to measure student learning.

Results

The analysis of the outcomes of the course suggests the training is effective in improving critical thinking in experimental design. The course was well received by the students as measured by student evaluations (Kirkpatrick Model Level 1). Improved scores on post-tests indicate that the students learned from the experience (Kirkpatrick Model Level 2). A template is provided for the implementation of similar courses at other institutions.

Conclusions

A problem-based learning platform appears effective in training newly matriculated graduate students in the required skills for designing experiments to test specific hypotheses, enhancing student preparation prior to initiation of their dissertation research.
Investigation of the milling characteristics of different focused-ion-beam sources assessed by three-dimensional electron diffraction from crystal lamellae
Authors: Parkhurst James M. , Crawshaw Adam D. , Siebert C. Alistair , Dumoux Maud , Owen C. David , Nunes Pedro , Waterman David , Glen Thomas , Stuart David I. , Naismith James H. , Evans Gwyndaf
Published: March 2023

Three-dimensional electron diffraction (3DED) from nanocrystals of biological macromolecules requires the use of very small crystals. These are typically less than 300 nm-thick in the direction of the electron beam due to the strong interaction between electrons and matter. In recent years, focused-ion-beam (FIB) milling has been used in the preparation of thin samples for 3DED. These instruments typically use a gallium liquid metal ion source. Inductively coupled plasma (ICP) sources in principle offer faster milling rates. Little work has been done to quantify the damage these sources cause to delicate biological samples at cryogenic temperatures. Here, an analysis of the effect that milling with plasma FIB (pFIB) instrumentation has on lysozyme crystals is presented. This work evaluates both argon and xenon plasmas and compares them with crystals milled with a gallium source. A milling protocol was employed that utilizes an overtilt to produce wedge-shaped lamellae with a shallow thickness gradient which yielded very thin crystalline samples. 3DED data were then acquired and standard data-processing statistics were employed to assess the quality of the diffraction data. An upper bound to the depth of the pFIB-milling damage layer of between 42.5 and 50 nm is reported, corresponding to half the thickness of the thinnest lamellae that resulted in usable diffraction data. A lower bound of between 32.5 and 40 nm is also reported, based on a literature survey of the minimum amount of diffracting material required for 3DED.