bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis ofS-adenosylmethionine-dependent Allosteric Transition from Active to Inactive States in Methylenetetrahydrofolate Reductase
Authors: Yamada Kazuhiro , Mendoza Johnny , Koutmos Markos
Published: December 2023

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and AdoMet-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures ofChaetomium thermophilumMTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form ofcMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites inhMTHFR, three of which are novel. Based on the structural framework provided by ourcMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.
Imputation approaches and quality standards for single-cell epigenetic age predictions
Authors: Liu Zian , Samee Md Abul Hassan
Published: December 2023

DNA methylation describes the addition of methyl groups, often between CpG dinucleotides. Single-cell bisulfite sequencing technologies allow the measurement of DNA methylation levels within individual cells. Epigenetic clocks are statistical models for computing biological age from DNA methylation levels, and have been used for detecting age variations in various disease contexts. However, there have been no attempts to apply epigenetic clocks to single-cell methylation data in humans. Thus, we questioned whether pre-built epigenetic clocks could be applied to single-cell methylation data; if so, how can we perform data quality control and imputation. We concluded that 1) linear regression-based epigenetic clocks can be applied to bisulfite-sequencing data, 2) data quality control can be used to reach the desired level of prediction accuracy, 3) first-principle imputation strategies could be used for missing data on selected CpG methylation sites, and 4) machine learning-based imputation tools could be used for accuracy-based age predictions. We built the first training-free, reference data-free framework for estimating epigenetic age in human single-cells, which would provide a foundation for future single-cell methylation-based age analyses.
Fructose-1-kinase has pleiotropic roles inEscherichia coli
Authors: Weeramange Chamitha , Menjivar Cindy , O’Neil Pierce T. , Qaidi Samir El , Harrison Kelly S. , Meinhardt Sarah , Bird Cole L. , Sreenivasan Shwetha , Hardwidge Philip R. , Fenton Aron W. , Hefty P. Scott , Bose Jeffrey L. , Swint-Kruse Liskin
Published: December 2023

InEscherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that theE. colienzyme fructose-1-kinase (FruK) can carry out its “reverse” reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruKstrain andfruKcomplementation show that FruK has a broader role in metabolism than fructose catabolism. The ΔfruKstrain also alters biofilm formation. SincefruKitself is repressed by Cra, these newly-reported events add layers to the dynamic regulation ofE. colicentral metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.
RNA-guided RNA silencing by an Asgard archaeal Argonaute
Authors: Bastiaanssen Carolien , Ugarte Pilar Bobadilla , Kim Kijun , Feng Yanlei , Finocchio Giada , Anzelon Todd A. , Köstlbacher Stephan , Tamarit Daniel , Ettema Thijs J. G. , Jinek Martin , MacRae Ian J. , Joo Chirlmin , Swarts Daan C. , Wu Fabai
Published: December 2023

Eukaryotic Argonaute proteins achieve gene repression and defense against viruses and transposons by RNA-guided RNA silencing. By contrast, known prokaryotic Argonautes adopt single-stranded DNA as guides and/or targets, leaving the evolutionary origin of RNA-guided RNA silencing elusive. Here, we show an evolutionary expansion of Asgard archaeal Argonautes (asAgos), including the discovery of HrAgo1 from the Lokiarchaeon ‘CandidatusHarpocratesius repetitus’ that shares a common origin with eukaryotic PIWI proteins. HrAgo1 exhibits RNA-guided RNA cleavagein vitroand RNA silencing in human cells. The cryo-EM structure of HrAgo1 combined with quantitative single-molecule experiments reveals that HrAgo1 possesses hybrid structural features and target binding modes bridging those of the eukaryotic AGO and PIWI clades. Finally, genomic evidence suggests that eukaryotic Dicer-like processing of double-stranded RNA likely emerged as a mechanism of generating guide RNA for asAgos prior to eukaryogenesis. Our study provides new insights into the evolutionary origin and plasticity of Argonaute-based RNA silencing.
A proteolytic AAA+ machine poised to unfold a protein substrate
Authors: Ghanbarpour Alireza , Sauer Robert T. , Davis Joseph H.
Published: December 2023

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.
Clouds, oases for airborne microbes – Differential metagenomics/ metatranscriptomics analyses of cloudy and clear atmospheric situations
Authors: Péguilhan Raphaëlle , Rossi Florent , Joly Muriel , Nasr Engy , Batut Bérénice , Enault François , Ervens Barbara , Amato Pierre
Published: December 2023

Bacteria cells and fungal spores can aerosolize and remain suspended in the atmosphere for several days, exposed to water limitation, oxidation, and lack of nutrients. Using comparative metagenomics/metatranscriptomics, we show that clouds are associated with the activation of numerous metabolic functions in airborne microorganisms, including fungal spore germination. The whole phenomenon mirrors the rapid recovery of microbial activity in soils after rewetting by rain, known as the “Birch effect”. Insufficient nutrient resources in cloud droplets cause a famine that recycling cellular structures could alleviate. The recovery of metabolic activity by microorganisms in clouds could favor surface invasion upon deposition, but it may also compromise further survival upon cloud evaporation. In any case, clouds appear as floating biologically active aquatic systems.

One-Sentence Summary

Clouds activate metabolic processes in airborne microorganisms
Assessing adverse effects and unspecific effects of transcutaneous spinal direct current stimulation (tsDCS)
Authors: Zhao Hongyan , Horn Ulrike , Freund Melanie , Bujanow Anna , Gundlach Christopher , Hartwigsen Gesa , Eippert Falk
Published: December 2023

Background

Transcutaneous spinal direct current stimulation (tsDCS) is a relatively recent method for non-invasively modulating neuronal activity in the human spinal cord. Despite its growing prominence, comprehensive studies addressing its potential adverse effects (AEs) and unspecific effects (UEs) are lacking.

Objective

In this study, we conducted a systematic investigation into the potential AEs and UEs of tsDCS in healthy volunteers.

Methods

We used a randomized double-blind within-participant design, employing anodal, cathodal and sham tsDCS of the thoracolumbar spinal cord. Our approach involved a newly-developed structured questionnaire (to assess subjectively-reported AEs) in combination with tsDCS-concurrent recording of skin conductance, cardiac and respiratory activity (to assess UEs in bodily state).

Results

The most frequently participant-reported AEs were sensations of burning, tingling, and itching, although they were largely described as mild; skin redness (experimenter-reported) occurred even more frequently. Importantly, when comparing AEs between active and sham tsDCS via frequentist and Bayesian analysis approaches, the results were largely in favour of no difference between conditions (with the exception of skin redness). A similar picture emerged for most UE metrics, suggesting that tsDCS does not induce changes in bodily state, at least as measured by our autonomic nervous system metrics.

Conclusion

We believe that the strategy employed here could serve as a starting point for a systematic AE and UE assessment in clinical populations, longitudinal designs and when stimulating different spinal sites. Taken together, our results contribute to assessing the tolerability, safety and specificity of tsDCS, in order to further the investigation of spinal cord function in health and disease.
Split GFP assay to show chloroplast targeting ofAgrobacteriumVirD2 protein
Authors: Matsuoka Aki , Ferranti Julia L. , Maliga Pal
Published: December 2023

Regenerating fertileArabidopsis thalianaplants from tissue culture cells with transformed plastid genomes is difficult, because of somaclonal variation in tissue culture cells. For nuclear gene transformation, tissue culture limitations were overcome in Arabidopsis by direct transformation of the female gametes using the floral dip protocol and identification of transgenic events in the seed progeny. DuringAgrobacteriumtransformation the VirD2 protein guides the T-complex, consisting of single stranded transferred-DNA (T-DNA) coated with VirE2 proteins, to the plant nucleus. To enable floral dip transformation of the plastid DNA, we retargeted VirD2 to chloroplasts. We show plastid targeting of VirD2 in a split GFP assay, where VirD2-GFP11complements GFP1-10in chloroplasts. Floral dip transformation of plastids will avoid tissue culture altogether, making plastid transformation readily available for the research community.
Uncovering Hidden Data in the AMNH Marine Invertebrate Collections
Authors: Wilkinson A. , Berniker L. , Johnson C. , Rodriguez E.
Published: December 2023

Average global sea surface temperatures have been consistently higher during the past thirty years than any other time since reliable temperature recordings in 1880. Marine life is sufficiently adaptable to survive in a range of temperatures, but extended periods of temperature extremes can have a profound effect on the biology and behavior of aquatic organisms. Historic baseline data is often critical to understanding these changes in biology, distribution, migration, and population dynamics. Data from museum specimens can provide such baselines, but are often an overlooked resource, in part because museum collection data are not readily available. The American Museum of Natural History’s invertebrate zoology collection is home to an estimated 7.5 million marine specimens dating back to the mid-1800s. Although many of the specimens have been catalogued in a public online database, data from a large number of these specimens are not yet in digital form and are therefore only accessible to staff and researchers visiting or borrowing from the collection. The Museum’s Crustacea collection contains around 19,400 specimen lots which hold significant historic value, including lots from the New York Zoological Society’s Bermuda Oceanographic Expeditions.

The Bermuda Oceanographic Expeditions provide a rare opportunity to examine historic abundance and distribution of pelagic crustacea taxa that are currently experiencing extended periods of increased ocean temperatures. We synthesized data from the fluid-preserved specimens and from expedition field logs to compose a depth profile that compared the number of crustacean specimens, identified to family or lower taxonomic level, per depth. The results indicate that crustacean abundance and diversity was highest (4-7 families) between 500-1000 fathoms. Most of the specimens were diel-migratory shrimp from the family Sergestidae. We believe that this study provides a useful basis of historic sergestid distribution data that can be used to contextualize modern research in the warming Sargasso Sea.
NAD: Noise-augmented direct sequencing of target nucleic acids by augmenting with noise and selective sampling
Authors: Shim Hyunjin
Published: December 2023

Next-generation sequencing necessitates a minimum quantity and concentration of DNA/RNA samples, typically achieved through amplification using the PCR technique. However, this amplification step introduces several drawbacks to biological insights, including PCR bias and the loss of epigenetic information. The advent of long-read sequencing technologies facilitates direct sequencing, with the primary constraint being the limited amount of DNA/RNA present in biological samples. Here, we present a novel method called Noise-Augmented Direct (NAD) sequencing that enables the direct sequencing of target DNA even when it falls below the minimum quantity and concentration required for long-read sequencing by augmenting with noise DNA and adaptive sampling. Adaptive sampling is an emerging technology of nanopore sequencing, allowing the enhanced sequencing of target DNA by selectively depleting noise DNA. In this study, we use the DNA standard of the Lambda phage genome as the noise DNA to augment samples containing low amounts of bacterial genomes (1 ng to 300 ng). The results with cost-effective flow cells indicate that NAD sequencing successfully detects the target DNA with an input quantity as low as 1 ng, and the bacterial genome ofSalmonella entericacan be assembled to 30% completion at an accuracy of 98% with an input quantity of 3 ng. With high throughput flow cells, the bacterial genome ofPseudonomas aeruginaswas assembled to near completion (99.9%) at an accuracy of 99.97% with an input quantity of 300 ng. This proof-of-concept study demonstrates the potential of NAD sequencing in enhancing the robustness of long-read sequencing with small input DNA/RNA samples with noise augmentation and adaptive sampling.
Engram Reactivation Mimics Cellular Signatures of Fear
Authors: Suthard Rebecca L. , Senne Ryan A. , Buzharsky Michelle D. , Diep Anh H. , Pyo Angela Y. , Ramirez Steve
Published: December 2023

Engrams, or the physical substrate of memory in the brain, recruit heterogeneous cell-types. Targeted reactivation of neurons processing discrete memories drives the behavioral expression of memory, though the underlying landscape of recruited cells and their real-time responses remain elusive. To understand how artificial stimulation of fear affects intra-hippocampal neuronal and astrocytic dynamics as well as their behavioral consequences, we expressed channelrhodopsin-2 in an activity-dependent manner in dentate gyrus neurons while performing fiber photometry of both cell types in ventral CA1 across learning and memory. Neurons and astrocytes were shock-responsive, while astrocytic calcium events were uniquely modulated by fear conditioning. Notably, optogenetic stimulation of a hippocampus-mediated engram recapitulated coordinated calcium signatures time-locked to freezing that were also observed during natural fear memory recall, suggesting that engram activation alters activity across different cell types within hippocampal circuits during the behavioral expression of fear. Together, our data reveals cell-type specific hippocampal dynamics during freezing behavior and points to neuronal-astrocytic coupling as a shared mechanism enabling the natural and artificial recall of a memory.

Highlights

Ventral hippocampal neurons and astrocytes are active during foot shock

Calcium activity is time-locked to freezing during fear conditioning and recall

Optogenetic reactivation of fear recapitulates cellular signatures seen during recall

Reactivation of a fear memory allows prediction of freezing behavior
HELZ promotes R loop resolution to facilitate DNA double-strand break repair by homologous recombination
Authors: Haji-Seyed-Javadi Ramona , Koyen Allyson E. , Rath Sandip K. , Madden Matthew Z. , Hou Yingzi , Song Boying S. , Kenney Anna M. , Lan Li , Yao Bing , Yu David S.
Published: December 2023

R loops are RNA-DNA hybrid containing structures involved in diverse cellular processes, including DNA double-strand break (DSB) repair. R loop homeostasis involving the formation and resolution of R loops is critical for DSB repair, and its dysregulation leads to genome instability. Here we show that the HELZ helicase promotes R loop resolution to facilitate DSB repair by homologous recombination (HR). HELZ depletion causes hypersensitivity to DSB-inducing agents, and HELZ localizes and binds to DSBs. HELZ depletion further leads to genomic instability in a R loop dependent manner and the accumulation of R loops globally and at DSBs. HELZ binds to R loops in response to DSBs and promotes their resolution, thereby facilitating HR to promote genome integrity. Our findings thus define a role for HELZ in promoting the resolution of R loops critical for DSB repair by HR.
The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity
Authors: Kalkreuter Edward , Kautsar Satria A. , Yang Dong , Bader Chantal D. , Teijaro Christiana N. , Fluegel Lucas L. , Davis Christina M. , Simpson Johnathon R. , Lauterbach Lukas , Steele Andrew D. , Gui Chun , Meng Song , Li Gengnan , Viehrig Konrad , Ye Fei , Su Ping , Kiefer Alexander F. , Nichols Angela , Cepeda Alexis J. , Yan Wei , Fan Boyi , Jiang Yanlong , Adhikari Ajeeth , Zheng Cheng-Jian , Schuster Layla , Cowan Tyler M. , Smanski Michael J. , Chevrette Marc G. , de Carvalho Luiz P. S. , Shen Ben
Published: December 2023

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ∼7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal’s strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.
Pan-azole- and multi-fungicide-resistantAspergillus fumigatusis widespread in the United States
Authors: Celia-Sanchez BN , Mangum B , Gómez Londoño LF , Wang C , Shuman B , Brewer MT , Momany M
Published: December 2023

Aspergillus fumigatusis an important global fungal pathogen of humans. Azole drugs are among the most effective treatments forA. fumigatusinfection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistantA. fumigatushas been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance inA. fumigatusare tandem repeats (TR) in thecyp51Apromoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistantA. fumigatushas been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistantA. fumigatusin the U.S., we collected isolates from agricultural sites in 8 states and tested 202 isolates for sensitivity to azoles. We found azole-resistantA. fumigatusin agricultural environments in 7 states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades and that TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow with clear recombination between two clades highlighting the potential for azole-resistance to continue spreading in the U.S.

Importance

Aspergillus fumigatusis a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris and compost. Azoles are the first line of defense antifungal drugs againstA. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistantA. fumigatushas been a problem in Europe and Asia for twenty years and has recently been reported in patients in the U.S. Until this study we didn’t know much about azole-resistantA. fumigatusin agricultural settings in the U.S. In this study we isolated azole-resistantA. fumigatusfrom multiple states and compared it to isolates from around the world. We show thatA. fumigatusthat is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.
In-vivoTherapeutic Evaluation of the Wound Healing: Potential of Ethanolic Extract ofTrichilia heudelotiiLeaves in Wistar Rats
Authors: Victoria Awolola Gbonjubola , Hassan Jinadu Taiwo , Toyin Yakubu Musa , Oyetunji Oyewopo Adeoye , Aborode Abdullahi Tunde , Ayobami Onifade Isreal
Published: December 2023

Background

Trichilia heudelotiibelongs to theMeliaceaefamily and is commonly used in indigenous African medicine to prevent and treat various microbial ailments.

Methods

Trichilia heudelotii’sethanolic leaf extracts contains phytochemicals which was studied to demonstrate its wound-healing properties. Phytochemical screening of leaf extracts contains flavonoids, phenolics, tannins, terpenoids, saponins, and alkaloids, which play a vital part in the healing process. Overall, quantitative, and qualitative screening were performed to evaluate the therapeutic efficacy ofTrichilia heudelotii’sphytochemicals for wound treatment.

Results

The wound-healing potency was evaluated using different concentrations of formulated oil extracts (0.5, 1.0, and 2.0g/10g) on fresh wounds inflicted on Wistar rats using the excision model. Healing capacity was assessed based on rate of wound contracting ability, time of wound closure, and epithelisation period. The results shows that leaf extract oils exhibited significant wound healing capacity (p<0.05) compared with controls. On the 18th day in the infected group using 2.0/10g extract formulated ointment, 100% wound closure was observed. On day 21, using 0.5/10g and 1.0/ 10g, extract formulated ointment compared with controls which exhibited 79% and 89%, respectively. In addition, histopathological characteristics show increased and well-organized new bands of collagen and fibroblasts in the extract formulated treatment than in the controls, which supports the wounding healing potential of the plant. This is the first report providing therapeutic evidence for the wound healing activity of ethanolic leaf extract on Wistar rats.

Conclusion

This indicate the evidence that establish the traditional use ofTrichilia heudelotiiextract for treating skin infections and wounds. The results also raise the possibility of developing a commercialized medicinal product-based venture based on this extract.

Graphical Abstract
Ecological and spatial overlap indicate interspecific competition during North American Canid radiation
Authors: Graciotti Rodolfo P. , Porto Lucas , Quental Tiago B.
Published: December 2023

Understanding biodiversity patterns and the processes that generate them are key goals in ecology and macroevolutionary studies. Diversity-dependent models of diversification have been used to indirectly infer the relevance of interspecific competition on driving speciation and extinction dynamics. In this study, we develop a new approach that more explicitly incorporated spatial and eco-morphological overlap among species to test how interspecific competition my affect diversification dynamics at deep time. We built different metrics that capture not only species temporal coexistence, but also their coexistence in space and morphospace to test the hypothesis that an increase in the intensity of competition would result in both a decrease in speciation rate and an increase in extinction rate. We tested our predictions using the North American fossil record of the family Canidae, a group that has been extensively studied and well characterized both from the eco-morphological and paleontological points of view. We found that interspecific competition only affected diversification dynamics during the early stages of Canidae radiation, resulting only in the suppression of speciation rate at the time the clade was expanding in diversity. We found no association between the intensity of the competition and extinction dynamics as expected by a competitive model, nor an association between changes in speciation and extinction rates and changes in global temperature, suggesting that extinction dynamics might be more related to external factors, such as clade-competition. We discuss the relevance of different factors on driving diversification dynamics changes over time and how evaluating the role of interspecific competition using different metrics that better capture the intensity of competition (as opposed to diversity dependent models), might be a way forward to investigate the role of biotic interactions at deep time.
Covalent Attachment of Horseradish Peroxidase to Single-Walled Carbon Nanotubes for Hydrogen Peroxide Detection
Authors: Ledesma Francis , Nishitani Shoichi , Cunningham Francis J. , Hubbard Joshua D. , Yim Dabin , Lui Alison , Chio Linda , Murali Aishwarya , Landry Markita P.
Published: December 2023

Single-walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near-infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre-existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Towards this end, we introduce a generalizable platform to generate protein-SWCNT-based optical sensors and use this strategy to synthesize a hydrogen peroxide (H2O2) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. We demonstrate a concentration-dependent response to H2O2, confirm the nanosensor can image H2O2in real-time, and assess the nanosensor’s selectivity for H2O2against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. We anticipate this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery.
Smart Parallel Automated Cryo Electron tomography
Authors: Eisenstein Fabian , Fukuda Yoshiyuki , Danev Radostin
Published: December 2023

In situcryo electron tomography enables investigation of macromolecules in their native cellular environment. Data collection, however, requires an experienced operator and valuable microscope time to carefully select targets for tilt series acquisition. Here, we developed a workflow using machine learning approaches to fully automate the entire process, including lamella detection, biological feature segmentation, target selection, and tilt series acquisition, all without the need for human intervention.
ORP9-PH domain-based fluorescent reporters for visualizing phosphatidylinositol 4-phosphate dynamics in living cells
Authors: Ajiki Moeka , Yoshikawa Masaru , Miyazaki Tomoki , Kawasaki Asami , Aoki Kazuhiro , Nakatsu Fubito , Tsukiji Shinya
Published: December 2023

Fluorescent reporters that visualize phosphatidylinositol 4-phosphate (PI4P) in living cells are indispensable to elucidate the roles of this fundamental lipid in cell physiology. However, currently available PI4P reporters have limitations, such as Golgi-biased localization and low detection sensitivity. Here, we present a series of fluorescent PI4P reporters based on the pleckstrin homology (PH) domain of oxysterol-binding protein-related protein 9 (ORP9). We show that the green fluorescent protein AcGFP1-tagged ORP9-PH domain can be used as a fluorescent PI4P reporter to detect cellular PI4P across its wide distribution at multiple cellular locations, including the plasma membrane (PM), Golgi, endosomes, and lysosomes with high specificity and contrast. We also developed blue, red, and near-infrared fluorescent PI4P reporters suitable for multicolor fluorescence imaging experiments. Finally, we demonstrate the utility of the ORP9-PH domain-based reporter to visualize dynamic changes in the PI4P distribution and level in living cells upon synthetic ER–PM membrane contact manipulation and GPCR stimulation. This work offers a new set of genetically encoded fluorescent PI4P reporters that are practically useful for the study of PI4P biology.
MFSD1 in complex with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes
Authors: Jungnickel Katharina Esther Julia , Guelle Océane , Iguchi Miharu , Dong Wentao , Kotov Vadim , Gabriel Florian , Debacker Cécile , Dairou Julien , McCort-Tranchepain Isabelle , Laqtom Nouf N. , Chan Sze Ham , Ejima Akika , Sato Kenji , Massa López David , Saftig Paul , Mehdipour Ahmad Reza , Abu-Remaileh Monther , Gasnier Bruno , Löw Christian , Damme Markus
Published: December 2023

Lysosomal degradation of macromolecules in lysosomes produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here, we deorphanized the Major Facilitator Superfamily Domain Containing 1 (MFSD1) protein, which forms a tight complex with the Glycosylated Lysosomal Membrane Protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer, and fluorescence-based studies inXenopusoocytes and proteoliposomes showed that MFSD1/GLMP acts as a uniporter for cationic and neutral dipeptides. Cryo-EM structure of the dipeptide-bound MFSD1/GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.
Systematic review and meta-analysis on the effects of chronic peri-adolescent cannabinoid exposure on schizophrenia-like behaviour in rodents
Authors: Li Zhikun , Mukherjee Diptendu , Duric Bea , Austin-Zimmerman Isabelle , Trotta Giulia , Spinazzola Edoardo , Quattrone Diego , Murray Robin M , Di Forti Marta
Published: December 2023

Background

The link between cannabis use and schizophrenia is well-established in epidemiological studies, especially among adolescents with early-onset use. However, this association in rodent models is less clear. This meta-analysis examined the effects of adolescent cannabinoid exposure on distinct schizophrenia-like behaviours in rodents and how experimental variations influence outcomes.

Methods

Following a pre-registered protocol (CRD42022338761), we searched PubMed, Ovid Medline, Embse and APA PsychInfo for English-language original studies until 2022. We synthesised data from experiments on schizophrenia-like behaviour in rats and mice after repeated peri-pubertal (onset between P23-P45) cannabinoid exposure. Risk of bias was assessed using the SYRCLE’s tool.

Results

We included 291 experiments from 91 articles across 9 behavioural tests. We found meta-analytic evidence supporting that CB1R agonists, both natural and synthetic, elicited broad schizophrenia-like behavioural alterations, including impaired working memory (g =-0.58 [CI: -1.00,-0.16]), novel object recognition (g=-0.63 [CI: -0.97,-0.30]), novel object location recognition (g=-0.70 [CI: -1.22,-0.28]), social motivation (g=-0.40 [CI: -0.63, -0.16]), pre-pulse inhibition (g=-0.48 [CI: -0.89, -0.08]), and sucrose preference (g=-0.92 [CI: -1.87,0.04]). By contrast, effects on novelty-induced locomotion were negligible. Subgroup analyses revealed similar effects across sexes and species. Substantial variance in the protocols and moderate-to-high heterogeneity in behavioural outcomes were observed. We found CBD may attenuate novelty-induced locomotion in an open field and enhance fear memory recall, but data was limited.

Discussion

This is the first meta-analysis to comprehensively assess the link between cannabinoids and schizophrenia-like behaviours in rodents. Our results support epidemiological links between early cannabis use and schizophrenia-like phenotypes, confirming the utility of animal models. Standardising protocols will optimise models to strengthen reproducibility and comparisons, our work provides a framework for refining rodent models to elucidate biological pathways linking cannabis and schizophrenia.
Molecular Epidemiology of multidrug-resistantKlebsiella pneumoniae, Enterobacter cloacae,andEscherichia colioutbreak among neonates in Tembisa Hospital, South Africa
Authors: Sekyere John Osei , Mmatli Masego , Bosch Anel , Ntsoane Ramathetje Virginia , Naidoo Harishia , Doyisa Sinenhlanhla , Maningi Nontuthuko E. , Mbelle Nontombi Marylucy , Said Mohamed
Published: December 2023

Background

An outbreak of multidrug-resistantKlebsiella pneumoniae, Escherichia coli, and Enterobacter cloacaeinfections in a neonatal ward within a tertiary hospital in South Africa resulted in the mortality of 10 patients within six months. In this work, the genomic epidemiology of and the molecular factors mediating this outbreak were investigated.

Methods

Bacterial cultures obtained from clinical samples collected from the infected neonates underwent phenotypic and molecular analyses to determine their species, sensitivity to antibiotics, production of carbapenemases, complete resistance genes profile, clonality, epidemiology, and evolutionary relationships. Mobile genetic elements flanking the resistance genes and facilitating their spread were also characterized.

Results

The outbreak was centered in two major wards and affected mainly neonates between September 2019 and March 2020. Most isolates (n = 27 isolates) wereK. pneumoniaewhile bothE. coliandE. cloacaehad three isolates each. Notably, 33/34 isolates were multidrug resistant (MDR), with 30 being resistant to at least four drug classes. All the isolates were carbapenemase-positive, but fourblaOXA-48isolates were susceptible to carbapenems.BlaNDM-1(n = 13) andblaOXA-48/181(n = 15) were respectively found onIS91 andIS6-likeIS26 composite transposons in the isolates alongside several other resistance genes. The repertoire of resistance and virulence genes, insertion sequences, and plasmid replicon types in the strains explains their virulence, resistance, and quick dissemination among the neonates.

Conclusions

The outbreak of fatal MDR infections in the neonatal wards were mediated by clonal (vertical) and horizontal (plasmid-mediated) spread of resistant and virulent strains (and genes) that have been also circulating locally and globally.
Low genetic variability and high isolation of a post-harvest South American pinniped population as revealed by genome-wide SNPs
Authors: Peralta Diego M. , Ibañez Ezequiel A. , Lucero Sergio , Cappozzo Humberto L. , Ceballos Santiago G. , Túnez Juan I.
Published: December 2023

Otaria flavescenshas been one of the most heavily exploited pinnipeds during the last 200 years with depletions of about 90% in some colonies. After the prohibition on sealing in South America, populations became stabilized except for the Uruguayan population, which showed a constant decrease. The underlying causes of its decline have remained unknown. This study used the RAD-seq approach to assess the variability and connectivity of some of the most overexploited sea lion colonies in the Atlantic Ocean. Our results revealed low allelic richness, nucleotide diversity and heterozygosity in the Uruguayan population and evidence of complete isolation from the Argentinean populations under study. In contrast, the Patagonian populations showed a high degree of connectivity, which could explain their recovery and high levels of current diversity. Our research emphasizes the precarious genetic status of the Uruguayan sea lion population, calling for the immediate implementation of conservation measures.
Dissociable effects of urgency and evidence accumulation during reaching revealed by dynamic multisensory integration
Authors: Hoffmann Anne H. , Crevecoeur Frédéric
Published: December 2023

When making perceptual decisions, humans combine information across sensory modalities dependent on their respective uncertainties. However, it remains unknown how the brain handles multisensory integration during movement, and which factors besides sensory uncertainty might influence the contribution of different modalities. We performed two reaching experiments on healthy adults to investigate whether movement corrections to combined visual and mechanical perturbations scale with visual uncertainty. To describe the dynamics of multimodal feedback responses, we further varied movement speed and duration of visual feedback during the movement. The results of our first experiment (N=16, 11 females) show that the contribution of visual feedback decreased with uncertainty. Interestingly, we observed a transient phase during which visual feedback responses were stronger during faster movements. In a follow-up experiment (N=16, 10 females), we found that the contribution of vision increased more quickly during slow movements when we presented the visual feedback for a longer time. Using an optimal feedback control model, we show that the increased response to visual feedback during fast movements can be explained by an urgency-dependent increase in control gains. Further, the fact that viewing duration increased the visual contributions suggests that the brain indeed performs a continuous state-estimation as expected in the optimal control model featuring a Kalman filter. Hence, both uncertainty and urgency determine how the sensorimotor system responds to multimodal perturbation during reaching control. We highlight similarities between reaching control and decision-making, both of which appear to be influenced by the accumulation of sensory evidence as well as response urgency.

Significance statement

The exact time course of multisensory integration during movement, along with the factors that influence this process, still requires further investigation. Here, we tested how visual uncertainty, movement speed, and visual feedback duration influence corrective movements during reaching with combined visual and mechanical perturbations. Using an optimal feedback control model, we illustrate that the time course of multimodal feedback responses follows the predictions of a Kalman filter which continuously weighs sensory feedback and internal predictions according to their reliability. Importantly, we further show that changes in movement speed led to urgency-dependent modulations of control gains. Our results highlight connections between motor control and decision-making processes, which both depend on the accumulation of sensory evidence and response urgency.
cAMP in budding yeast: a messenger for sucrose metabolism?
Authors: Botman Dennis , Kanagasabapathi Sineka , Rep Mila I. , van Rossum Kelly , Tutucci Evelina , Teusink Bas
Published: December 2023

S. cerevisiae(or budding yeast) is an important micro-organism for sucrose-based fermentation in biotechnology. Yet, it is largely unknown how budding yeast adapts to sucrose transitions. Sucrose can only be metabolized when the invertase or the maltose machinery are expressed and we propose that the Gpr1p receptor signals extracellular sucrose availability via the cAMP peak to adapt cells accordingly. A transition to sucrose or glucose gave a transient cAMP peak which was maximally induced for sucrose. When transitioned to sucrose, cAMP signalling mutants showed an impaired cAMP peak together with a lower growth rate, a longer lag phase and a higher yield compared to a glucose transition. These effects were not caused by altered activity or expression of enzymes involved in sucrose metabolism and imply a more general metabolic adaptation defect. Basal cAMP levels were comparable among the mutant strains, suggesting that the transient cAMP peak is required to adapt cells correctly to sucrose. We propose that the short-term dynamics of the cAMP signalling cascade detects long-term extracellular sucrose availability and speculate that its function is to maintain a fermentative phenotype at continuously low glucose and fructose concentrations.
Development and characterization of a non-human primate model of disseminated synucleinopathy
Authors: Rico Alberto J. , Corcho Almudena , Chocarro Julia , Ariznabarreta Goiaz , Roda Elvira , Honrubia Adriana , Arnaiz Patricia , Lanciego José L.
Published: December 2023

The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. In brief, cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyrus in particular), together with neurons located in the caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.
What pupil size can and cannot tell about math anxiety
Authors: Blini Elvio , Anobile Giovanni , Arrighi Roberto
Published: December 2023

Math Anxiety (MA)consists of excessive fear and worry about math-related situations. It represents a major barrier to numerical competence and the pursuit of STEM careers. Yet it is still poorly evaluated, mostly through self-reports. Here we sought to probePupil Size (PS)as a viable biomarker of MA by administering arithmetic problems to young adults (N= 70) with various levels of MA. We found that arithmetic competence and performance are indeed negatively associated with MA, and this is accurately tracked by PS. When performance is accounted for, MA does not further modulate PS (before, during, or after calculation). However, the latency of PS peak dilation could add a significant contribution to predicting MA scores, indicating that high MA may be accompanied by more prolonged cognitive effort. Results show that MA and mathematical competence may be too crystalized in young adults to be discernible, calling for early educational interventions.
AAV-mediated interneuron-specific gene replacement for Dravet syndrome
Authors: Mich John K. , Ryu Jiyun , Wei Aguan D. , Gore Bryan B. , Guo Rong , Bard Angela M. , Martinez Refugio A. , Bishaw Yemeserach , Luber Em , Oliveira Santos Luiz M. , Miranda Nicole , Ramirez Jan-Marino , Ting Jonathan T. , Lein Ed S. , Levi Boaz P. , Kalume Franck K.
Published: December 2023

Dravet syndrome (DS) is a devastating developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10-20% rate of premature death. Most DS patients harbor loss-of-function mutations in one copy ofSCN1A, which has been associated with inhibitory neuron dysfunction. Here we developed an interneuron-targeting AAV humanSCN1Agene replacement therapy using cell class-specific enhancers. We generated a split-intein fusion form ofSCN1Ato circumvent AAV packaging limitations and deliverSCN1Avia a dual vector approach using cell class-specific enhancers. These constructs produced full-length NaV1.1 protein and functional sodium channels in HEK293 cells and in brain cellsin vivo. After packaging these vectors into enhancer-AAVs and administering to mice, immunohistochemical analyses showed telencephalic GABAergic interneuron-specific and dose-dependent transgene biodistribution. These vectors conferred strong dose-dependent protection against postnatal mortality and seizures in two DS mouse models carrying independent loss-of-function alleles ofScn1a,at two independent research sites, supporting the robustness of this approach. No mortality or toxicity was observed in wild-type mice injected with single vectors expressing either the N-terminal or C-terminal halves ofSCN1A, or the dual vector system targeting interneurons. In contrast, nonselective neuronal targeting ofSCN1Aconferred less rescue against mortality and presented substantial preweaning lethality. These findings demonstrate proof-of-concept that interneuron-specific AAV-mediatedSCN1Agene replacement is sufficient for significant rescue in DS mouse models and suggest it could be an effective therapeutic approach for patients with DS.