bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphetamine increases timing variability by degrading prefrontal ramping activity
Authors: Weber Matthew A. , Sivakumar Kartik , Kirkpatrick Braedon Q. , Stutt Hannah R. , Tabakovic Ervina E. , Bova Alexandra S , Kim Young-cho , Narayanan Nandakumar S.
Published: September 2024

Background

Amphetamine is a commonly abused psychostimulant that increases synaptic catecholamine levels and impairs executive functions. However, it is unknown how acute amphetamine affects brain areas involved in executive control, such as the prefrontal cortex. We studied this problem in mice using interval timing, which requires participants to estimate an interval of several seconds with a motor response. Rodent prefrontal cortex ensembles are required for interval timing. We tested the hypothesis that amphetamine disrupts interval timing by degrading prefrontal cortex temporal encoding.

Methods

We first quantified the effects of amphetamine on interval timing performance by conducting a meta-analysis of 11 prior rodent studies. We also implanted multielectrode recording arrays in the dorsomedial prefrontal cortex of 7 mice and then examined the effects of 1.5 mg/kgD-amphetamine injected intraperitoneally on interval timing behavior and prefrontal neuronal ensemble activity.

Results

A meta-analysis of previous literature revealed that amphetamine produces a large effect size on interval timing variability across studies but only a medium effect size on central tendencies of interval timing. We found a similar effect on interval timing variability in our task, which was accompanied by greater trial-to-trial variability in prefrontal ramping, attenuated interactions between pairs of ramping neurons, and dampened low-frequency oscillations.

Conclusions

These findings suggest that amphetamine alters prefrontal temporal processing by increasing the variability of prefrontal ramping. Our work provides insight into how amphetamine affects timing-related brain activity, which may be useful in developing new neurophysiological markers for amphetamine use and novel treatments targeting the prefrontal cortex.
Rhythmic Sampling and Competition of Target and Distractor in a Motion Detection Task
Authors: Xiong Changhao , Petro Nathan M. , Bo Ke , Cui Lihan , Keil Andreas , Ding Mingzhou
Published: September 2024

It has been suggested that the visual system samples attended information rhythmically. Does rhythmic sampling also apply to distracting information? How do attended information and distracting information compete temporally for neural representations? We recorded electroencephalography from participants who detected instances of coherent motion in a random dot kinematogram (RDK; the target stimulus), overlayed on different categories (pleasant, neutral, and unpleasant) of affective images from the International Affective System (IAPS) (the distractor). The moving dots were flickered at 4.29 Hz whereas the IAPS pictures were flickered at 6 Hz. The time course of spectral power at 4.29 Hz (dot response) was taken to index the temporal dynamics of target processing. The spatial pattern of the power at 6 Hz was similarly extracted and subjected to a MVPA decoding analysis to index the temporal dynamics of processing pleasant, neutral, or unpleasant distractor pictures. We found that (1) both target processing and distractor processing exhibited rhythmicity at ∼1 Hz and (2) the phase difference between the two rhythmic time courses were related to task performance, i.e., relative phase closer to π predicted a higher rate of coherent motion detection whereas relative phase closer to 0 predicted a lower rate of coherent motion detection. These results suggest that (1) in a target-distractor scenario, both attended and distracting information were sampled rhythmically and (2) the more target sampling and distractor sampling were separated in time within a sampling cycle, the less distraction effects were observed, both at the neural and the behavioral level.
Microfluidic Modulation of Microvasculature in Microdissected Tumors
Authors: Nguyen Tran N. H. , Horowitz Lisa F. , Nguyen Brandon , Lockhart Ethan , Zhu Songli , Gujral Taranjit S. , Folch Albert
Published: September 2024

The microvasculature within the tumor microenvironment (TME) plays an essential role in cancer signaling beyond nutrient delivery. However, it has been challenging to control the generation and/or maintenance of microvasculature inex vivosystems, a critical step for establishing cancer models of high clinical biomimicry. There have been great successes in engineering tissues incorporating microvasculaturede novo(e.g., organoids and organs-on-chip), but these reconstituted tissues are formed with non-native cellular and molecular components that can skew certain outcomes such as drug efficacy. Microdissected tumors, on the other hand, show promise in preserving the TME, which is key for creating cancer models that can bridge the gap between bench and bedside. However, microdissected tumors are challenging to perfuse. Here, we developed a microfluidic platform that allows for perfusing the microvasculature of microdissected tumors. We demonstrate that, compared to diffusive transport, microfluidically perfused tissues feature larger and longer microvascular structures, with a better expression of CD31, a marker for endothelial cells, as analyzed by 3D imaging. This study also explores the effects of nitric oxide pathway-related drugs on endothelial cells, which are sensitive to shear stress and can activate endothelial nitric oxide synthase, producing nitric oxide. Our findings highlight the critical role of controlled perfusion and biochemical modulation in preserving tumor microvasculature, offering valuable insights for developing more effective cancer treatments.
Efficient degradation of oxidized polyethylene containing ester bonds by cutinase or lipase in aqueous phase
Authors: Kong Demin , Wang Lei , Xia Wei , Shi Meng , Wu Jing
Published: September 2024

Polyethylene is a widely used plastic that presents challenges in degrading within natural environments. Therefore, the degradation of polyethylene is of utmost importance. Currently, physical or chemical methods are efficient but more dangerous. Conversely, biological methods are safer but less efficient. Combining the two approaches to create entirely new polyethylene degradation pathways is a viable idea. Based on the previous research, it is an effective method to add ester bonds inside polyethylene and degrade using cutinase or lipase. However, the method only detected a decrease in molecular weight and did not observe a rate of weight loss at the macroscopic level. In this research, rapid degradation of polyethylene is achieved by optimizing catalysis conditions and machine learning. Finally, the maximum weight loss up to 80.51% within 24h and a mild catalysis environment (60-80°C). Furthermore, the method’s versatility was confirmed for cutinase and lipase. Overall, this research has established an effective, efficient, and mild degradation process of polyethylene, significantly contributing to the progress in polyethylene degradation.
Field-testedHaHB11andHaHB4soybean exhibit increased grain number and heat tolerance at the reproductive stage
Authors: Raineri Jesica , Montero Bulacio Enrique , Campi Mabel , Portapila Margarita , Otegui María Elena , Chan Raquel Lía
Published: September 2024

Soybean is one of the primary sources of vegetable oil and protein worldwide. However, its yield improvement has lagged behind the other major crops. This study explored the potential of the sunflower transcription factor HaHB11 to enhance soybean yield and heat stress tolerance. We generated transgenic soybean plants expressing HaHB11 and evaluated their performance across four field trials. The HaHB11 plants showed a significant increase in grain number per plant compared to controls, which can be related to an increased number of nodes and pods per plant. Flowering dynamics analysis revealed delayed blooming and an increased number of flowers per node, leading to a higher pod set, particularly between nodes four and six. Principal component analysis across field trials identified temperature as a crucial factor influencing grain number, enhancing the differences exhibited by HaHB11 plants. The pollen from transgenic plants germinated better, and tubes were longer than controls under heat stress. Carbohydrate distribution analyses indicated differential allocation of nutrients, supporting the increased pod and grain set in HaHB11 plants. Additionally, vegetation indices can distinguish HaHB11 plants from controls in several developmental stages. These results indicated that HaHB11 enhances soybean yield under heat stress, becoming a promising technology for soybean improvement.

Highlight

Soybean transformed with the sunflower geneHaHB11was tested in the field for four campaigns, showing differential allocation of nutrients, increased number of nodes, pods, grains, and heat tolerance.
Burden of Latent Tuberculosis Infection among Healthcare Workers of Primary Health Centres and its Association with Clinical Laboratory Parameters
Authors: Selvavinayagam Sivaprakasam T. , Sankar Ganga , Yong Yean K. , Sankar Sathish , Zhang Ying , Tan Hong Y. , Balakrishnan Pachamuthu , Murugesan Amudhan , Rajeshkumar Manivannan , Frederick Asha , Kumar Masilamani Senthil , PriyaRaj Paulkanraj , Prabhakaran Jesudoss , Sangeetha Pattusamy , Arunpathy Pasupathy , Charu Rajamani , Muruganandam Nagarajan , Sakate Deepak M. , Jayakumar Deepak , Dhandapani Prabu , Rudrapathy Parthiban , Velu Vijayakumar , Emmenegger Marc , Larsson Marie , Shankar Esaki M. , Raju Sivadoss
Published: September 2024

Objectives

Healthcare workers (HCWs) are at high risk of latent tuberculosis infection (LTBI) due to their continued occupational exposure. The incidence of LTBI among the HCWs has seldom been investigated. Primary health centres (PHCs) provide effective and affordable medical care largely for the local population. The HCWs of PHCs who are likely to have increased occupational exposure have an increased risk of reactive as well as LTBI, and contribute to overall TB transmission than the general population.

Methods

A cross-sectional study (March–April 2024) was carried out to assess the burden of LTBI among the HCWs of PHCs (n=64) across Chennai, India. Blood samples were analyzed for gamma interferon using a QuantiFERON-TB Gold Plus assay. A complete baseline health profile, including complete blood count, blood glucose, liver and renal functions, and ferritin levels was estimated to identify potential biomarkers that are independently associated with LTBI.

Results

The present study revealed an LTBI prevalence of 25.3% (n=99) among PHC workers. The red cell distribution width was significantly associated with LTBI positivity among different biochemical and haematological parameters analyzed. Factors such as individuals’ age, underlying comorbid conditions (30.3%; n=30), and longer employment duration (28%; n=28) were significantly associated with IGRA positivity. IGRA positivity was significantly associated with decreased RDW-SD in females, overweight, and participants with ‘O’ blood group.

Conclusions

The study reported a high prevalence of LTBI among HCWs of PHCs, which necessitates their periodical screening for the elimination of TB.
Structural Characterization of mRNA Lipid Nanoparticles in the Presence of Intrinsic Drug-free Lipid Nanoparticles
Authors: Chen Xiaoxia , Ye Yongfeng , Li Mengrong , Zuo Taisen , Xie Zhenhua , Ke Yubin , Cheng He , Hong Liang , Liu Zhuo
Published: September 2024

Lipid nanoparticles (LNPs) have emerged as a versatile platform for mRNA delivery across a range of applications, including disease prevention, cancer immunotherapy, and gene editing. Structural models of mRNA-containing lipid nanoparticles (mRNA-LNPs) have also been proposed based on characterization of samples by using various advanced techniques. Among these, small angle neutron scattering (SANS) has proven essential for elucidating the lipid distribution within mRNA-LNPs, a factor crucial to both their preparation and efficacy. However, recent findings suggest that the mRNA-LNP samples prepared via commercial microfluidic techniques may contain a substantial fraction of drug-free LNPs, casting doubt on the validity of earlier structural models. In this study, we employed contrast variation SANS to characterize both drug-free LNPs and our mRNA-LNP sample, and quantified the proportion of drug-free LNPs present to be ∼30% in our mRNA-LNP sample using nano flow cytometry. By removing the contributions of drug-free LNPs from the SANS data of our mRNA-LNP sample, we were able to precisely characterize the structure of mRNA-LNPs. Consequently, we proposed structural models for both drug-free LNPs and mRNA-LNPs. Notably, our analysis revealed similar lipid distributions and shell thicknesses between the two particle types, while the solvent content in mRNA-LNPs was significantly higher, leading to a larger core size. This work not only offers a method for accurately characterizing the structure of mRNA-LNPs, but also establishes criteria for selecting appropriate analytical techniques based on the structural parameters of interest. Therefore, our findings hold significant implications for the mechanistic understanding and quality control of mRNA-based vaccines.

Significance

Precise structural determination of mRNA-containing lipid nanoparticles (mRNA-LNPs) is vital for mechanistic insights into their preparation, delivery, immunogenicity, and storage, which are critical to the development of mRNA-based vaccines. However, most previous studies overlooked the substantial presence of drug-free LNPs within these samples. Here, we identified that approximately 30% of the nanoparticles in our mRNA-LNP sample were drug-free. By integrating contrast variation small angle neutron scattering (SANS) data from both drug-free LNPs and mRNA-LNPs, we developed structural models for both particle types, and provided a guidance for characterization technique selection based on concerned structural features. Beyond mechanistic insight on structure, our approach offers a robust method for quality assessment and process monitoring in mRNA-based vaccine production.
Measuring 3-dimensional dynamics of membrane in living cell with metal-induced energy transfer imaging/spectroscopy
Authors: Chen Tao , Gallea José Ignacio , Karedla Narain , Wang Dongxia , Enderlein Jörg
Published: September 2024

The dynamics of cellular membranes are crucial for many essential cellular processes. Despite various methods available to study membrane dynamics, simultaneously measuring membrane diffusion and fluctuations within living cell membranes remains challenging. In this study, we developed a method combining metal-induced energy transfer (MIET) with fluorescence correlation spectroscopy (FCS) to accurately measure the three-dimensional (3D) dynamics of membranes, including transverse diffusion and vertical fluctuations. Our approach achieves a spatiotemporal resolution of one nanometer and few microseconds. We demonstrate the efficacy of our technique through experiments on the diffusion and transversal undulations of model membranes. Additionally, we showcase the versatility of MIET-FCS by applying it to various membrane systems within living cells, such as the plasma membrane, endoplasmic reticulum, and nuclear envelope. Our findings provide a powerful tool for investigating complex membrane dynamics in diverse cellular environments, advancing the field of membrane biophysics.
Quantifying evolution of SNPs that affect RNA secondary structure inArabidopsis thalianagenes
Authors: Martin Galen T. , Fiscus Christopher J. , Gaut Brandon S.
Published: September 2024

Single-stranded RNA molecules have the ability to form intramolecular bonds between nucleotides and create secondary structures. These structures can have phenotypic effects, meaning mutations that alter secondary structure may be subject to natural selection. Here we examined the population genetics of these mutations withinArabidopsis thalianagenes. We began by identifying derived SNPs with the potential to alter secondary structures within coding regions, using a combination of computational prediction and empirical data analysis. We then examined nucleotide diversity and allele frequencies of these “pair-changing mutations” (pcM) in 1,001A. thalianagenomes to infer selective pressures on these sites. The pcM SNPs at synonymous sites had an 8.8% reduction in nucleotide diversity relative to non-pcM SNPs at synonymous sites, and they were found at lower allele frequencies. We used demographic modeling to estimate selection coefficients from the pcM allele frequency spectrum, finding that pcMs had estimated selection coefficients ∼3 to 5x smaller than nonsynonymous mutations. Additionally, we explored associations of pcMs with bioclimatic variables and gene expression. They exist at higher frequencies in higher latitudes and colder environments, with mean annual temperature explaining 51% of the variation in pcM frequency among subpopulations. Alleles containing pcMs had an average reduction of 137.4 normalized counts compared to genes with the ancestral secondary structure (mean expression = 3215.7 normalized counts). Overall, we conclude that structure-altering mutations are subject to intermediate levels of selection.
Evolution of tRNA pool shapes variation in selection on codon usage across the Saccharomycotina subphylum
Authors: Cope Alexander L. , Shah Premal
Published: September 2024

Across the major taxonomical domains, synonymous codons of an amino acid are found to be used in unequal frequencies. This codon usage bias – both in terms of the degree of bias and the identity of codons used – is highly variable, even among closely related species. Within a species, genome-wide codon usage bias reflects a balance between adaptive and non-adaptive microevolutionary processes. Variation in these microevolutionary processes results in across-species variation in codon usage bias. As codon usage bias is tightly linked to important molecular and biophysical processes, it is critical to understand how changes to these processes drive changes to the microevolutionary processes. Here we employ a population genetics model of coding sequence evolution to quantify natural selection and mutation biases on a per-codon basis and estimate gene expression levels across the budding yeasts Saccharomycotina subphylum. We interrogate the impact of variation in molecular mechanisms hypothesized to be driving the microevolution of codon usage. We find that natural selection and mutation biases evolved rapidly over macroevolutionary time, with high variability between closely related species. The majority (324/327) of yeasts exhibited clear signals of translational selection, with selection coefficients being correlated with codon-specific estimates of ribosome waiting times within species. Across species, natural selection on codon usage correlated with changes to ribosome waiting times, indicating that tRNA pool evolution is a major factor driving changes to natural selection on codon usage. We find evidence that changes to tRNA modification expression can contribute to changes in natural selection across species independent of changes to tRNA gene copy number, suggesting tRNA modifications also play a role in shaping natural selection on codon usage. Our work firmly establishes how changes to microevolutionary processes can be driven by changes to molecular mechanisms, ultimately shaping the macroevolutionary variation of a trait.
Abnormal vasculature reduces overlap between drugs and oxygen in a tumour computational model: implications for therapeutic efficacy
Authors: Enjalbert Romain , Köry Jakub , Krüger Timm , Bernabeu Miguel O.
Published: September 2024

The tumour microvasculature is abnormal, and as a consequence oxygen and drug transport to the tumour tissue is impaired. The abnormal microvasculature contributes to tumour tissue hypoxia, as well as to varying drug penetration depth in the tumour. Many anti-cancer treatments require the presence of oxygen to be fully efficacious, however the question of how well oxygen concentration overlaps with drug concentration is not elucidated, which could compromise the therapeutic effect of these drugs. In this work we use a computational model of blood flow and oxygen transport, and develop a model for an oxygen-dependent drug, T-DM1, to study the overlap of oxygen and drug concentration in healthy and tumour tissue, where we assume the tumour tissue to compress blood vessels. Our results show that, due to the compressed vessels present in tumours, areas of sufficient oxygen concentration for a drug to function overlap poorly with areas of sufficient drug concentration, covering 28% of the tumour tissue, compared to 82% in healthy tissue. The reduction in drug and oxygen overlap is due to the altered red blood cell dynamics through the abnormal microvasculature, and indicates that drug transport to tumours should not be considered independently of oxygen transport in cases where the drug requires oxygen to function.
Inter-kingdom signaling by theLegionellaautoinducer LAI-1 involves the antimicrobial guanylate binding protein GBP
Authors: Solger Franziska , Rauch Jonas , Vormittag Simone , Fan Mingzhen , Raykov Lyudmil , Charki Paul , Soldati Thierry , Seibel Jürgen , Hilbi Hubert
Published: September 2024

The causative agent of Legionnaires’ disease,Legionella pneumophila, is an amoebae-resistant environmental bacterium, which replicates intracellularly in a distinct compartment, the “Legionella-containing vacuole” (LCV).L. pneumophilaemploys the α-hydroxyketone compound LAI-1 (Legionellaautoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 promotes intracellular replication and inhibits the migration of mammalian cells andDictyostelium discoideum. In this study, we revealed that LAI-1 and “clickable” azido-LAI-1 derivatives inhibit the migration ofD. discoideumand localize to LCVs. Azido-LAI-1 colocalizes with the LCV markers calnexin, P4C, and AmtA, but not with mitochondrial or lipid droplet markers. Intriguingly, LAI-1 dependent inhibition ofD. discoideummigration involves the single guanylate-binding protein (GBP), a member of the GBP family of large GTPases, which in metazoan organisms promote cell autonomous immunity.D. discoideumlacking GBP (Δgnbp) allows more efficient intracellular replication ofL. pneumophila, without apparently compromising LCV remodeling or integrity, and GBP-GFP localizes to the ER at LCV-ER membrane contact sites (MCS). However, the peri-LCV localization of LAI-1 and GBP is not mutually dependent. Synthetic LAI-1 inhibits the expansion/remodeling of LCVs (but not vacuoles harboring avirulentL. pneumophila) in a GBP-dependent manner. Taken together, the work shows that LAI-1 localizes to LCVs, and LAI-1-dependent inter-kingdom signaling involvesD. discoideumGBP, which localizes to LCV-ER MCS and acts as an antimicrobial factor by restricting the intracellular growth ofL. pneumophila.

Author Summary

Small molecule inter-kingdom signaling between pathogens and host cells represents a crucial but only partly understood aspect of microbial virulence. The amoeba-resistant opportunistic pathogenLegionella pneumophilaemploys the compound LAI-1 (Legionellaautoinducer-1) for intra-species and inter-kingdom signaling. In metazoan cells, the conserved and wide-spread family of guanylate-binding protein (GBP) large GTPases usually comprises several distinct paralogues, which are implicated in pathogen detection, inflammation, cell death pathways, and cell autonomous immunity. In the social amoebaDictyostelium discoideum, only a singleGBPgene of unknown function is present. Using approaches from organic chemistry, genetics, cell biology and infection biology, we reveal that GBP is involved in the inhibition ofD. discoideummigration and pathogen vacuole expansion/remodeling by LAI-1 as well as in intracellular growth ofL. pneumophila. This study provides a novel link between small molecule inter-kingdom signaling and GBP-dependent cell autonomous immunity.
Extracellular Ligand-Responsive Translational Regulation of Synthetic mRNAs using Engineered Receptors
Authors: Nakanishi Hideyuki , Itaka Keiji
Published: September 2024

mRNA drugs can encode any protein and are regarded as a promising therapeutic modality. In this study, we developed a new mRNA system allowing extracellular ligand-responsive translational regulation. This system consists of three mRNAs, two of which encode components for detecting extracellular ligands, and the other encodes the protein of interest, with a binding motif on 5’UTR for translational regulation. In the presence of ligand biomolecules such as arginine vasopressin (AVP) and prostaglandin E2 (PGE2), the translation level of the protein of interest is upregulated or downregulated in a ligand biomolecule concentration-dependent manner. We showed upregulation of anti-inflammatory signaling in response to an inflammatory mediator, PGE2, demonstrating that this system allowed production of the therapeutic proteins according to the disease site environment. This system will pave the way for the next-generation mRNA drugs that self-adjust their protein production levels in response to fluctuating disease states.

Graphical Abstract

mRNAs encoding regulatory components (an engineered receptor and a translational regulator) and a target mRNA are co-transfected into mammalian cells. In the absence of the ligand, the target mRNA is translationally repressed by the translational regulator. The ligand induces engineered receptor-mediated degradation of the translational regulator protein, resulting in the translational activation of the target mRNA.
AAV9-mediated targeting of natural antisense transcript as a novel treatment for Dravet Syndrome
Authors: Diaz Juan Antinao , Chilcott Ellie , Almacellas Barbanoj Amanda , Keegan Anna , McTague Amy , Cross J Helen , Schorge Stephanie , Lignani Gabriele , Waddington Simon Nicholas , Karda Rajvinder
Published: September 2024

Dravet syndrome (DS) is a severe childhood onset developmental and epileptic encephalopathy which leads to life-long disability. Symptoms usually manifest in the first year of life and include prolonged severe seizures, developmental delay and severe intellectual disability. DS patients have an increased mortality rate, including sudden unexpected death in epilepsy (SUDEP). Approximately 90% of patients carry a heterozygous loss-of-function mutation in theSCN1Agene, which encodes a voltage-gated sodium ion channel, NaV1.1. The NaV1.1 channel is expressed in the brain and at a lower level, in the heart. Previous studies have identified a long non-coding RNA (lncRNA) which specifically downregulatesSCN1Agene expression. This natural antisense transcript (NAT) can be modulated by AntagoNATs, small synthetic oligonucleotides developed to inhibit NAT function. In a DS mouse model, AntagoNATs were shown to modulateScn1aexpression by targeting theScn1aNAT, improving seizure frequency after repeated administration. Here, we have developed novel AntagoNATs and incorporated these into a clinically relevant adeno-associated virus serotype 9 (AAV9) gene therapy vector, to test in a DS mouse model (Scn1a+/-) and provide a one-off treatment approach.

Eighteen AntagoNATs were testedin vitro; from the best performing candidates, we selected two AntagoNAT sequences (K & H) forin vivotesting as they had the highest homology (90%) to humanSCN1ANAT. We administered both vectors to newbornScn1a+/-mice via intracerebroventricular (ICV) and intravenous (IV) injection to target the brain and heart. AAV9-AntagoNAT-H significantly increased survival, decreased febrile seizures and reduced spontaneous seizure frequency compared to the PBS control group. When administered at P14 by ICV and IV injection, AAV9-AntagoNAT-H increased survival. In this proof-of-concept study, we have demonstrated for the first time the delivery of AntagoNAT technology via an AAV9 vector and thus offering the possibility of a one-time treatment for DS patients.
Time-resolved transcriptomic of singleV. viniferafruits: membrane transports as switches of the double sigmoidal growth
Authors: Savoi Stefania , Shi Mengyao , Sarah Gautier , Weber Audrey , Torregrosa Laurent , Romieu Charles
Published: September 2024

By revealing that the grape berry loses one H+per accumulated sucrose at the inception of ripening, adopting a single fruit paradigm elucidates the fundamentals of the malate-sugar nexus, previously obscured by asynchrony in population-based models of ripening. More broadly, the development of the individual fruit was revisited from scratch to capture the simultaneous changes in gene expression and metabolic fluxes in a kinetically relevant way from flowering to overripening. Dynamics in water, tartrate, malate, hexoses, and K+fluxes obtained by combining individual single fruit growth and concentration data allowed to define eleven sub-phases in fruit development, which distributed on a rigorous curve in RNAseq PCA. WGCNA achieved unprecedented time resolutions in exploring transcript level-metabolic rate associations. A comprehensive set of membrane transporters was found specifically expressed during the first growth phase related to vacuolar over-acidification. Unlike in slightly more acidic citrus, H+V-PPase transcripts were predominantly expressed, followed by V-ATPase and PH5, clarifying the thermodynamic limit beyond which replacement by the PH1/PH5 complex turns compulsory. Puzzlingly,bona fideALMT kept a low profile at this stage, possibly replaced by a predominating uncharacterized anion channel. Then, the switch role of HT6 in sugar accumulation was confirmed, electroneutralized by malate vacuolar leakage and H+pumps activation.

Highlights

To alleviate asynchronicity biases, transcripts showing strict coincidental timing with pericarp physiological phases were disentangled on single berries, enlightening the tight multifaceted membrane developmental control of sugar and acid fluxes.
A computationally designed panel of diverse and selective peroxygenases for terpene oxyfunctionalization
Authors: Münch Judith , Soler Jordi , Gildor-Cristal Ofir , Fleishman Sarel J. , Garcia-Borràs Marc , Weissenborn Martin J.
Published: September 2024

Enzyme engineering has a critical role in the transition to economical, low-energy and environmentally friendly chemical production. Current approaches relying on costly iterations of mutation and selection are limited to reactions with a straightforward experimental readout and struggle to address mutational epistasis. We focus on unspecific peroxygenases (UPOs), prized engineering targets due to their ability to oxyfunctionalize diverse organic molecules of industrial and environmental value. To address the lack of scalable screening for UPO functions, we applied enzyme-design calculations to focus experiments. Starting from an AlphaFold2 model ofMthUPO, the automated FuncLib algorithm generated 50 diverse active-site multipoint designs—all of which were functional. Screening against nine diverse terpenes revealed large improvements and new oxyfunctionalization products, resulting in molecules of high pharmaceutical and industrial value. We rationalized observed specificity changes using AI-based docking and molecular dynamics simulations, providing molecular insights that could generalize to engineering other UPOs. Thus, computational design and modeling can dramatically accelerate the urgently needed green transition of the chemical industry.

Abstract Figure
Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells
Authors: Uguen Mélanie , Shell Devan J. , Silva Madhushika , Deng Yu , Li Fengling , Szewczyk Magdalena M. , Yang Ka , Zhao Yani , Stashko Michael A. , Norris-Drouin Jacqueline L. , Waybright Jarod M. , Beldar Serap , Rectenwald Justin M. , Mordant Angie L. , Webb Thomas S. , Herring Laura E. , Arrowsmith Cheryl H. , Ackloo Suzanne , Gygi Steven P. , McGinty Robert K. , Barsyte-Lovejoy Dalia , Liu Pengda , Halabelian Levon , James Lindsey I. , Pearce Kenneth H. , Frye Stephen V.
Published: September 2024

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a kinact/KIof 1.0 x 106M-1s-1and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression.
What we can find in what’s left behind: DNA metabarcoding of semi-aquatic insect exuviae
Authors: Dworatzek SL , Tozer M , Austin K , Vermey A , Steinke D
Published: September 2024

Semi-aquatic insects contribute to critical ecological functioning of freshwater habitats in both the aquatic and terrestrial phase of their life. Traditional methods of studying their emergence rely on the capture of insects as they emerge, and morphological identification with taxonomic keys. This is not only time consuming but can have large impacts on the study population, obstacles that can be removed by the use of DNA.

This study investigated the potential of using exuviae collected from the water surface as DNA source. Both emergence trap samples and insect exuviae were collected from a constructed wetland and a small creek in southern Ontario. Metabarcoding provided a total of 40 samples with 254 distinct taxa from emergence traps (26 samples), and 135 from exuviae (14 samples). There were many similarities between both two sample types, especially for the rich chironomid diversity. Nonetheless, the exuviae samples were able to identify more orders containing semi-aquatic insects. Furthermore, they showed a higher level of diversity within these orders. This higher level of diversity seen in exuviae samples may be due to limitations of emergence traps, such as they only account for a small defined surface area. In contrast, exuviae are representing a much larger area and are free floating, thus collection of emerging taxa is not limited to the emerging site.

We were able to show that identification of emerging aquatic insects through metabarcoding of exuviae is a useful method for the study of insect emergence.
PEG Adjustment Enables Genome Transplantation UsingMycoplasma MycoidesRecipient
Authors: Karas Bogumil J. , Moreau Nicolette G. , Venter J Craig , Smith Hamilton O , Glass John I.
Published: September 2024

Pioneering advances in synthetic biology, initiated at the J. Craig Venter Institute, have enabled the creation of the first cell driven by synthetic genome and opened a new era of creating designer microbes. This technology offers far greater potential for genetic modification than traditional genome editing techniques and holds promise for the routine creation of synthetic cells as DNA synthesis costs decrease and genetic knowledge expands. Two essential technologies underpin this achievement: the ability to clone entire genomes in host organisms, such asSaccharomyces cerevisiae, and the successful transplant of these genomes into recipient cells to generate living organisms. In all previous work the recipient cell in genome transplantation experiments has beenMycoplasma capricolum. In this study, we explored the potential of usingMycoplasma mycoidesstrains as recipient cells for genome transplantation. By increasing polyethylene glycol (PEG) concentration from 5% to 10%, we successfully transplanted variousM. mycoidesstrains using severalM. mycoidesrecipient cells. Additionally, we demonstrated the ability to transplantM. capricolumgenomes intoM. mycoidesrecipient cells; although with lower efficiency compared toM. mycoidesstrains. These findings provide a modified transplantation protocol and likely get us closer to expanding genome transplantation to other bacterial species.
Intrinsically disordered proteins can behave as different polymers across their conformational ensemble
Authors: Chakraborty Saikat , Morozova Tatiana I. , Barrat Jean-Louis
Published: September 2024

Intrinsically disordered proteins (IDPs) are macromolecules, which in contrast to well-folded proteins, explore a large number of conformationally heterogeneous states. In this work, we investigate the conformational space of the disordered proteinβ-casein using Hamiltonian replica exchange atomistic molecular dynamics simulations in explicit water. The energy landscape contains a global minimum along with two shallow funnels. Employing static polymeric scaling laws separately for individual funnels, we find that they cannot be described by the same polymeric scaling exponent. Around the global minimum, the conformations are globular, whereas in the vicinity of local minima we recover coil-like scaling. To elucidate the implications of structural diversity on equilibrium dynamics, we initiate standard molecular dynamics simulations in the NVT ensemble with representative conformations from each funnel. Global and internal motions for different classes of trajectories show heterogeneous dynamics with globule to coil-like signatures. Thus, IDPs can behave as entirely different polymers in different regions of the conformational space.

TOC Graphic
Minocycline enhances antimicrobial activity of unpolarized macrophages againstAcinetobacter baumanniiwhile reducing the inflammatory response
Authors: Guerra Alberto Daniel , Volk Cecilia F. , Peterson-Weber Alex , Peters Jason M. , Sakoulas George , Sauer John-Demian , Rose Warren E.
Published: September 2024

Minocycline activity againstAcinetobacter baumannii(AB)in vivois underestimated by standard methods of susceptibility testing. We examined pharmacologic effects of minocycline on primary immunity that may be contributing to thein vivovs.in vitrodiscrepancy of minocycline activity against AB. Minocycline MICs against 10ABstrains were compared in standard bacteriologic media (Mueller-Hinton broth, MHB) and physiologic (RPMI) media. Macrophages were pretreated with minocycline or comparator antibiotics beforeABco-culture. Macrophage cytokine production and phagocytosis ofABwere measured without and with pre-treatment with minocycline. Two to eight-fold reduction in minocycline MIC against 10ABstrains occurred in RPMI compared to MHB, which was more pronounced than other antibiotic classes. Macrophages pretreated with 1, 5, 10, 30, 50, and 100 μg/mL minocycline before bacterial co-cultures significantly decreasedABinoculum at 6 hours of co-culture in a dose-dependent manner, with no bacterial colonies observed from co-cultures with macrophages pretreated with 30 μg/mL or more of minocycline. Macrophages pretreated with minocycline for 24 hours before zymosan stimulation led to significantly higher levels of phagocytosis. Macrophages treated with minocycline for 24 hours significantly decreased production of IL-6, TNF-α, and MCP-1 in a dose dependent manner. The minocyclinein vivoefficacy may be attributed to enhanced activity in nutrient-limited, physiologic medium combined with increased macrophage phagocyte efficiency. Incorporating novel assays that recapitulate thein vivoenvironment will be important for understanding the host-pathogen-antibiotic relationship toward a goal of improved future drug discovery and overall treatment strategies againstABand other drug-resistant pathogens.
Fast hierarchical processing of orthographic and semantic parafoveal information during natural reading
Authors: Wang Lijuan , Frisson Steven , Pan Yali , Jensen Ole
Published: September 2024

Readers extract orthographic and semantic information from parafoveal words before fixating on them. While this has to be achieved within an intersaccadic interval, the neuronal mechanisms supporting this fast parafoveal word processing within the language network remain unknown. We co-registered MEG and eye-tracking data in a natural reading paradigm to uncover the neuronal mechanisms supporting parafoveal processing. Representational similarity analysis (RSA) revealed that parafoveal orthographic neighbours (e.g., “writer” vs. “waiter”) showed higher representational similarity than non-neighbours (e.g., “writer” vs. “police”), emerging ∼68 ms after fixation onset on the preceding word (e.g., “clever”) in the visual word form area. Similarly, parafoveal semantic neighbours (e.g., “writer” vs. “author”) exhibited increased representational similarity at ∼137 ms in the left inferior frontal gyrus. Importantly, the degree of orthographic and semantic parafoveal processing predicted individual reading speed. Our findings suggest fast hierarchical processing of parafoveal words across distinct brain regions, which enhances reading efficiency.
Modelling trypanosome motility
Authors: Overberg Florian A. , Jamshidi Khameneh Narges , Krüger Timothy , Engstler Markus , Gompper Gerhard , Fedosov Dmitry A.
Published: September 2024

African trypanosomiasis, or sleeping sickness, is a life-threatening disease caused by the protozoan parasiteTrypanosoma brucei. The bloodstream form ofT. bruceihas a slender body with a relatively long active flagellum, which makes it an excellent swimmer. We develop a realistic trypanosome model and perform mesoscale hydrodynamic simulations to study the importance of various mechanical characteristics for trypanosome swimming behavior. The cell body is represented by an elastic triangulated network, while the attached flagellum consists of four interconnected running-in-parallel filaments, which permits a good control of the flagellum beating plane. Our simulation results are validated against experimental observations, and highlight the crucial role of body elasticity, non-uniform actuation along the flagellum length, and the orientation of flagellum-beating plane with respect to the body surface for trypanosome locomotion. These results offer a framework for exploring parasite behavior in complex environments.
Impact of AIM2 on HNSCC Development
Authors: Reinartz Dakota M. , Escamilla-River Vicente , Tribble Stephanie L. , Caulin Carlos , Wilson Justin E.
Published: September 2024

Head and neck squamous cell carcinoma (HNSCC) constitutes 90% of head and neck cancers. HNSCC development is linked to chronic inflammation, while established HNSCC tumors are often immune suppressive. However, both occur through mechanisms that are not fully understood. The cytosolic double-stranded DNA sensor Absent in Melanoma 2 (AIM2) is an inflammasome forming protein that also has inflammasome-distinct roles in restricting tumorigenesis by limited PI3K signaling. Here, we used an experimental mouse model of HNSCC, involving treatment of wild type (WT) andAim2−/−mice with the carcinogen 4NQO in drinking water. Compared to WT mice, 4NQO-treatedAim2−/−mice exhibited larger tumor sizes and increased tissue dysplasia. 4NQO-treated wild type andAim2−/−mice displayed similar tongueIl6, Tnf, Il1b, Il12, and Il10expression and no consistent differences in PI3K or inflammasome activation, suggesting AIM2 may not regulate these factors during HNSCC. Instead,IfngandIrf1was elevated in 4NQO-treatedAim2−/−mice, suggesting AIM2 restricts IFNγ. In line with this, RNA-sequencing of total tongue RNA from 4NQO-treated mice revealedAim2−/−mice had enhanced expression of genes related to the MHC protein complex, cell killing, and T cell activation compared to wild type mice. In addition, we observed increased macrophage infiltration into the tongue epithelium of 4NQO-treatedAim2−/−mice. Lastly, usingAim2−/−/Rag1−/−-double deficient animals, we found that the adaptive immune compartment was necessary for the enhanced tumorigenesis during AIM2 deficiency. Taken together, these findings suggest AIM2 limits the progression of oral tumor development partially through regulating IFNγ and adaptive immune responses.
Mechanism of allosteric activation in human mitochondrial ClpP protease
Authors: Goncalves Monica M. , Uday Adwaith B. , Forrester Taylor J. B. , Currie S. Quinn W. , Kim Angelina S. , Feng Yue , Jitkova Yulia , Velyvis Algirdas , Harkness Robert W. , Kimber Matthew S. , Schimmer Aaron D. , Zeytuni Natalie , Vahidi Siavash
Published: September 2024

Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement.

Significance statement

Human ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.