bluuebunny/crossref_metadata_2025 huggingface dataset:
Revisit the Inhibitory Effects of Glucocorticoids on Immunocytes
Authors: Wu Shuting , Zhao Shushu , Zhong Yiwei , Wang Bin
Published: January 2023

Glucocorticoids (GCs) are efficacious agents for reducing inflammation and suppressing immune responses, exerting various effects on immune cells through the intracellular glucocorticoid receptor (GR), and impacting both innate and adaptive immunity. In the context of COVID-19, glucocorticoids are often used to treat severe cases of patients by reducing inflammation, suppressing immune responses, and ameliorating the severity of COVID-19. However, the precise inhibitory effects on immune cells have yet to be comprehensively delineated. In this study, we extensively examined the inhibitory effects of treating Balb/c mice with dexamethasone (DEX) on lymphoid and myeloid cells. We observed that high doses of DEX treatment resulted in a reduction in the number of immunocytes and an attenuation of their activity. Particularly noteworthy, macrophages, DC cells, and monocytes were diminished by approximately 90% following high doses of DEX, while B cells experienced a reduction of about 70% and CD3 T cells were less affected. Furthermore, our findings demonstrated that DEX induces the inhibition of immune cells by engaging in high-affinity binding to GR. Consequently, we conclude that DEX treatments affect a broad range of immune cells, encompassing both lymphoid and myeloid cells, through depletion or the down-regulation of immune function, potentially acting via the GR signaling pathway. These findings may enhance the clinical applicability of DEX in achieving transient immune deficiency.
Trophic eggs affect caste determination in the antPogonomyrmex rugosus
Authors: Genzoni E. , Schwander T. , Keller L.
Published: January 2023

Understanding how a single genome creates distinct phenotypes remains a fundamental challenge for biologists. Social insects provide a striking example of polyphenism, with queen and worker castes exhibiting morphological, behavioural, and reproductive differences. Here we show that trophic eggs, which do not contain an embryo and are primarily regarded as a source of food, play a role in the process of caste differentiation in the harvester antPogonomyrmex rugosus. When first instar larvae were given access to trophic eggs, they mostly developed into workers. By contrast, larvae without access to trophic eggs developed into queens. We found that trophic eggs differ in many ways from viable eggs, including texture, morphology and their contents of protein, triglycerides, glycogen, sugar and small RNAs. Moreover, comparison of miRNA fragment size distributions suggests differences in the composition of miRNAs between the two egg types. This is the first demonstration of trophic eggs playing a role in caste differentiation in social insects.
Explainable prediction of catalysing enzymes from reactions using multilayer perceptrons
Authors: Probst Daniel
Published: January 2023

Assigning or proposing a catalysing enzyme given a chemical or biochemical reaction is of great interest to life sciences and chemistry alike. The exploration and design of metabolic pathways or the challenge of finding more sustainable enzyme-catalysed alternatives to traditional organic reactions are just two examples of tasks that require an association between reaction and enzyme. However, given the lack of large and balanced annotated data sets of enzyme-catalysed reactions, assigning an enzyme to a reaction still relies on expert-curated rules and databases. Here, we present a data-driven explainable human-in-the-loop machine learning approach to support and ultimately automate the association of a catalysing enzyme with a given biochemical reaction. In addition, the proposed method is capable of predicting enzymes as candidate catalysts for arbitrary organic reactions. Finally, the introduced explainability and visualisation methods can easily be generalised to support other machine-learning approaches involving chemical and biochemical reactions.
Live cell imaging and CLEM reveals effects of Mutant Huntingtin Aggregation Process
Authors: Yi̇ği̇t Esra Nur , Aydin Mehmet Şerif , Aksan Kurnaz Işıl , Eroğlu Emrah , Öztürk Gürkan
Published: January 2023

Huntington’s disease is a neurodegenerative disease caused by expansion of CAG repeats in exon-1 ofHuntingtin (HTT)gene leading to production of mutant Huntingtin (mHtt) protein. Htt protein is known to play crucial roles in regulation of cytoskeletal dynamics and vesicular transport in physiological conditions. By combiningin vitrotime-lapse imaging and correlative light and electron microscopy (CLEM), we investigated the subcellular dynamics of mHtt during the process aggregate formation. Here we show that distribution of F-actin is affected by mHtt aggregation. F-actin tends to relocate from the peripheral to perinuclear area in cells with mHtt aggregates, possibly caused by sequestration of F-actin from cell membrane to aggregation zones. In accordance with this, mHtt in hippocampal neurons were aggregated into axonal varicosities together with F-actin as they have higher F-actin expression in their neurites compared to their soma. Additionally, correlative light and electron microscopy (CLEM) revealed increased mitochondrial accumulation at the periphery of mHtt aggregates which is surrounded by a F-actin mesh. Mitochondria targeted HyPerRed, a genetically encoded hydrogen peroxide sensor, revealed that oxidative stress in mHtt expressing cells increased. Thus, our findings give new insights about the pathology caused by mHtt through interplay of aggregates with F-actin cytoskeleton and mitochondrial oxidative stress.
α-synuclein preformed fibrils bind to β-neurexins and impair β-neurexin-mediated presynaptic organization
Authors: Feller Benjamin , Fallon Aurélie , Luo Wen , Nguyen Phuong Trang , Shlaifer Irina , Lee Alfred Kihoon , Karkout Samer , Bourgault Steve , Durcan Thomas M , Takahashi Hideto
Published: January 2023

Synucleinopathies form a group of neurodegenerative diseases defined by misfolding and aggregation of alpha-synuclein (α-syn). Abnormal accumulation and spreading of α-syn aggregates lead to synapse dysfunction and neuronal cell death. Yet, little is known about synaptic mechanisms underlying α-syn pathology. Here we identified β-isoforms of neurexins (β-NRXs) as presynaptic organizing proteins that interact with α-syn preformed fibrils (α-syn PFFs), toxic α-syn aggregates, but not α-syn monomers. Our cell surface protein binding assays and surface plasmon resonance assays reveal that α-syn PFFs bind directly to β-NRX through their N-terminal histidine-rich domain (HRD) at nanomolar range (Kd: ~500 nM monomer equivalent). Furthermore, our artificial synapse formation assays show that α-syn PFFs diminish excitatory and inhibitory presynaptic organization induced by a specific isoform of neuroligin 1 that binds only β-NRXs, but not α-isoforms of neurexins. Thus, our data suggest that α-syn PFFs interact with β-NRXs to inhibit β-NRX-mediated presynaptic organization, providing novel molecular insight into how α-syn PFFs induce synaptic pathology in synucleinopathies such as Parkinson’s disease and dementia with Lewy bodies.
Network analysis reveals strain-dependent response to misfolded tau aggregates
Authors: Acri Dominic J. , You Yanwen , Tate Mason D. , McCord Brianne , Sharify A. Daniel , John Sutha , Karahan Hande , Kim Byungwook , Dabin Luke C. , Philtjens Stéphanie , Wijeratne H.R. Sagara , McCray Tyler J. , Smith Daniel C. , Bissel Stephanie J. , Lamb Bruce T. , Lasagna-Reeves Cristian A. , Kim Jungsu
Published: January 2023

Mouse genetic backgrounds have been shown to modulate amyloid accumulation and propagation of tau aggregates. Previous research into these effects has highlighted the importance of studying the impact of genetic heterogeneity on modeling Alzheimer’s disease. However, it is unknown what mechanisms underly these effects of genetic background on modeling Alzheimer’s disease, specifically tau aggregate-driven pathogenicity. In this study, we induced tau aggregation in wild-derived mice by expressingMAPT(P301L). To investigate the effect of genetic background on the action of tau aggregates, we performed RNA sequencing with brains of 6-month-old C57BL/6J, CAST/EiJ, PWK/PhJ, and WSB/EiJ mice (n=64). We also measured tau seeding activity in the cortex of these mice. We identified three gene signatures: core transcriptional signature, unique signature for each wild-derived genetic background, and tau seeding-associated signature. Our data suggest that microglial response to tau seeds is elevated in CAST/EiJ and PWK/PhJ mice. Together, our study provides the first evidence that mouse genetic context influences the seeding of tau.

Graphical Abstract

Seeding of tau predates the phosphorylation and spreading of tau aggregates. Acri and colleagues report transcriptomic responses to tau and elevated tau seeds in wild-derived mice. This paper creates a rich resource by combining genetics, tau biosensor assays, and transcriptomics.
Too bad to be fraud, Midjourney has yet to embark in science
Authors: Cammer Michael
Published: January 2023

Artificial Intelligence (AI) promises great benefits for data management, for image analysis, and for new discovery, but also creates unprecedented potential for fraud. This brief study explores whether images created by AI pose a threat to the integrity of figures in scientific publications. An attempt was made to use a leading AI image-generation engine, Midjourney, to create plausible facsimiles of images that are commonly depicted in manuscripts in the biological sciences. We conclude that a widely available AI for generated images is not a threat to integrity, yet, but may be in the near future.
Changes in biodiversity impact atmospheric chemistry and climate through plant volatiles and particles
Authors: Sanaei Anvar , Herrmann Hartmut , Alshaabi Loreen , Beck Jan , Ferlian Olga , Fomba Khanneh Wadinga , Haferkorn Sylvia , van Pinxteren Manuela , Quaas Johannes , Quosh Julius , Rabe René , Wirth Christian , Eisenhauer Nico , Weigelt Alexandra
Published: January 2023

Climate extremes in tandem with biodiversity change affect emissions of biogenic volatile organic compounds (BVOCs) from plants and, as a result, the formation of biogenic secondary organic aerosols (BSOA). The resulting BSOA can have a wide variety of impacts, such as on Earth’s radiative balance or cloud- and precipitation formation. However, at present, it is unclear how changing biodiversity will lead to changes in BVOC emissions, BSOA formation and their corresponding effects. We present a conceptual framework of the relationships between biodiversity and BVOC emissions based on our current mechanistic understanding and combining knowledge from the fields of biology and atmospheric chemistry. Parts of this framework are tested in a case study using a tree diversity experiment with adjunct BVOC and BSOA characterisation. The relative differences in tree monocultures and mixtures show that the overall concentration of BVOCs decreases with increasing biodiversity (p< 0.01), but results for BSOA compounds are mixed and overall non-significant (p= 0.40). We suggest future studies should follow a multidisciplinary approach where the fields of biology, atmospheric chemistry and climate research interact.
Duck pan-genome reveals two transposon-derived structural variations caused bodyweight enlarging and white plumage phenotype formation during evolution
Authors: Wang Kejun , Hua Guoying , Li Jingyi , Yang Yu , Zhang Chenxi , Yang Lan , Hu Xiaoyu , Scheben Armin , Wu Yanan , Gong Ping , Zhang Shuangjie , Fan Yanfeng , Zeng Tao , Lu Lizhi , Gong Yanzhang , Jiang Ruirui , Sun Guirong , Tian Yadong , Kang Xiangtao , Hu Haifei , Li Wenting
Published: January 2023

Structural variations (SVs) are a major source of domestication and improvement traits, however SV profiles of duck and their phenotypic impacts largely hidden. We present the first duck pan-genome constructed using five genome assemblies capturing ∼40.98 Mb new sequences. This pan-genome together with high-depth sequencing data (∼46.5X) identified 101,041 SVs, of which substantial proportions were derived from transposable element (TE) activity. Many TE-derived SVs anchoring in a gene body or regulatory region are linked to duck’s domestication and improvement. By combining quantitative genetics with molecular experiments, we dissect how TE-derived SVs change gene expression ofIGF2BP1and generate a novel transcript ofMITF, shaping bodyweight and white plumage. In theIGF2BP1locus, the TE-derived SV explains the largest effect on bodyweight among avian species (27.61% of phenotypic variation). Our findings highlight the importance of using a pan-genome as a reference in genomics studies and explore the roles of TE-derived SVs in trait formation and in livestock breeding.
m6A Demethylase FTO Stabilizes LINK-A to Exert Oncogenic Roles via MCM3-Mediated Cell Cycle Progression and HIF-1α Activation
Authors: Nan Yabing , Liu Shi , Luo Qingyu , Wu Xiaowei , Zhao Pengfei , Chang Wan , Liu Zhihua
Published: January 2023

RNAN6-methyladenosine (m6A) modification, balanced by methyltransferases and demethylases, has recently been shown to play critical roles in multiple cancers. However, the mechanism by which m6A modification regulates long noncoding RNA (lncRNA) stability and function during cancer progression remains unclear. Here, we show that m6A demethylase fat mass and obesity-associated protein (FTO) removes the m6A modification on long intergenic noncoding RNA for kinase activation (LINK-A) and stabilizes it to promote cell proliferation and cytotoxic chemotherapy resistance in esophageal squamous cell carcinoma (ESCC). Mechanistically, LINK-A enhances the interaction between minichromosome maintenance complex component 3 (MCM3) and cyclin-dependent kinase 1 (CDK1) to promote MCM3 phosphorylation by CDK1. MCM3 is a subunit of the hexameric protein complex and its phosphorylation facilitates loading of the MCM complex onto chromatin, which promotes cell cycle progression and subsequent cell proliferation. Meanwhile, LINK-A prevents the interaction of MCM3 and hypoxia-inducible factor 1α (HIF-1α), abrogates MCM3-mediated transcriptional repression of HIF-1α, and promotes glycolysis and chemoresistance of cancer cells. These results elucidate a mechanism whereby FTO-stabilized LINK-A plays oncogenic roles and present the FTO/LINK-A/MCM3/HIF-1α axis as a promising therapeutic target for ESCC.
Frequency and machine learning predictors of depressive, anxiety, obsessive-compulsive symptoms, and suicidal ideation among university students
Authors: Meda Nicola , Pardini Susanna , Rigobello Paolo , Visioli Francesco , Novara Caterina
Published: January 2023

Introduction

Prospective studies on mental health of students showed that young adults enroled in university are affected by poorer mental health than other working peers or adults, and this condition is responsible for a large proportion of disability-adjusted life-years

Methods

We enrolled 1388 students at the baseline (and 557 completed follow-up after six months) who reported their demographic information and completed self-report questionnaires on depressive, anxiety and obsessive-compulsive symptoms. We applied multiple regression modelling and supervised machine learning to evince associations and predict the risk factors of poorer mental health at baseline and follow-up

Results

Approximately one out of five students reported severe depressive symptoms and/or suicidal ideation. An association of economic worry with depression was evidenced at the beginning of the study (when there was a high frequency of worry OR = 3.11 [1.88 – 5.15]) and during follow-up. Supervised machine learning exhibited high accuracy in predicting the students who maintained well-being (balanced accuracy = 0.85) or absence of suicidal ideation, but its performance was almost null in identifying those whose symptoms worsened.

Conclusions

Students’ severe mental health problems are reaching worrying percentages, and few demographic factors can be leveraged to predict poor mental health outcomes. Further research including people with lived experience is crucial to assess students’ needs and improve the prediction of those at risk of developing worse symptoms.
Probable transmission of SARS-CoV-2 from an African lion to zoo employees
Authors: Siegrist Audrey A. , Richardson Kira L. , Ghai Ria R. , Pope Brian , Yeadon Jamie , Culp Betsy , Behravesh Casey Barton , Liu Lixia , Brown Jennifer A. , Boyer Leslie V.
Published: January 2023

Animal to human transmission of SARS-CoV-2 has not previously been reported in a zoo setting. A vaccinated African lion with physical limitations requiring hand feeding tested positive for SARS-CoV-2 after development of respiratory signs. Zoo employees were screened, monitored prospectively for development of symptoms, then re-screened as indicated, with confirmation by RT-PCR and whole-genome virus sequencing when possible. Trace-back investigation narrowed the source of infection to one of five people. Three exposed employees subsequently developed symptoms, two with viral genomes identical to the lion’s. Forward contact tracing investigation confirmed probable lion-to-human transmission.

Close contact with large cats is a risk factor for bidirectional zoonotic SARS-CoV-2 transmission that should be considered when occupational health and biosecurity practices at zoos are designed and implemented. SARS-CoV-2 rapid testing and detection methods in big cats and other susceptible animals should be developed and validated to facilitate timely implementation of One Health investigations.
Structural Elucidation of β1- and β2-Transferrin Using Microprobe-Capture In-Emitter Elution and High-Resolution Mass Spectrometry
Authors: Luo Ruben Yiqi , Pfaffroth Christopher , Yang Samuel , Hoang Kevin , Yeung Priscilla S.-W. , Zehnder James L. , Shi Run-Zhang
Published: January 2023

Background

Cerebrospinal fluid (CSF) leak is typically diagnosed by detecting a protein marker β2-transferrin (β2-Tf) in secretion samples. β2-Tf and β1-transferrin (β1-Tf) are glycoforms of human transferrin (Tf). A novel affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to analyze the Tf glycoforms and elucidate the structures of β1-Tf and β2-Tf.

Methods

To implement MPIE, an analyte is first captured on the surface of a microprobe, and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via next-generation biolayer interferometry (BLI). When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for HR-MS analysis. Serum, CSF, and secretion samples were analyzed using MPIE-ESI-MS.

Results

Based on the MPIE-ESI-MS results, the structures of β1-Tf and β2-Tf were solved. As Tf glycoforms, β1-Tf and β2-Tf share the amino acid sequence but have varying N-glycans. β1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611. β2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611.

Conclusions

The structures of β1-Tf and β2-Tf were successfully elucidated by MPIE-ESI-MS analysis. The resolving power of the novel MPIE-ESI-MS method was demonstrated in this study. On the other hand, knowing the N-glycan structures on β2-Tf allows for the design of other novel test methods for β2-Tf in the future.
Improved Ethanol Tolerance and Production in Pyruvate Dehydrogenase Mutant ofSaccharomyces cerevisiae
Authors: Yang Anna , Ranaivoarisoa Tahina O. , Bose Arpita
Published: January 2023

Ethanol, a naturally synthesized compound bySaccharomyces cerevisiaeyeast through alcoholic fermentation, has previously been studied as a renewable alternative to traditional fossil fuels. However, current challenges of engineeringS. cerevisiaestrains for ethanol production remain: low ethanol productivity, inefficient substrate catabolism, and a buildup of toxic products to inhibitory levels. In this study, we proposed a method of metabolic rewiring via the deletion of thepda1gene, which leads to pyruvate dehydrogenase (PDH) deficiency. The Δpda1mutant strain was created by CRISPR Cas-9 knockout using the constructed pCRCT-PDA1 plasmid. Subsequently, mutant candidates were screened by PCR and Sanger sequencing, confirming a 17 bp deletion in thepda1gene. The wild-type and mutant strains were analyzed for growth under aerobic and anaerobic conditions in glucose and glycerol, as well as ethanol production and tolerance. The Δpda1mutant displays a ~two-fold increase in anaerobic ethanol production and an aerobic growth defect with no observed increase in ethanol production. The mutant is also hyper-tolerant to ethanol, which allows a faster buildup of products in growth media with minimal reduction in growth. This newS. cerevisiaestrain deficient in PDH may provide a solution to the efficient and abundant synthesis of biofuels such as ethanol by redirecting metabolic flux and altering stress response.
Salience-based information integration: An overarching function of the “social brain”?
Authors: Lugrin Claire , Konovalov Arkady , Ruff Christian
Published: January 2023

Behavior in social contexts is routinely accompanied by neural activity in a brain network comprising the bilateral temporoparietal junction (TPJ), dorsomedial and dorsolateral prefrontal cortex (dmPFC and dlPFC), and precuneus. This network – often referred to as the “social brain network” (SBN) – is thought to have evolved in response to the information processing demands of life in social groups. However, its precise functional contributions to behavior are unclear, since many of its areas are also activated in non-social contexts requiring, for example, attentional orienting or context updating. Here we argue that these results may reflect a basic neural mechanism implemented by areas in this network that is commonly required in both social and non-social contexts: Integrating multiple sensory and memory inputs into salient configurations, such as social constellations or perceptual Gestalts. We tested this hypothesis using a numeracy paradigm that orthogonally varied the salience of sensory target configurations and the required motor responses. Even in this non-social task, several regions of the SBN (TPJ, dmPFC, and precuneus) showed higher activity when the goal required the brain to attend to more versus less salient perceptual configurations. This activation pattern was specific to configuration salience and did not reflect general task demand or switching to new contexts. Taken together, these results suggest that the integration of information into salient configurations may be a key function of SBN regions, thus offering a new perspective on the widespread recruitment of these areas across social and non-social contexts.
Reciprocal regulation of TLR4, TLR3 and Macrophage Scavenger Receptor 1 regulates nonopsonic phagocytosis of the fungal pathogenCryptococcus neoformans
Authors: Onyishi Chinaemerem U. , Desanti Guillaume E. , Wilkinson Alex L. , Fejer Gyorgy , Christophe Olivier D. , Bryant Clare E. , Mukhopadhyay Subhankar , Gordon Siamon , May Robin C.
Published: January 2023

The opportunistic fungal pathogenCryptococcus neoformanscauses lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear howC. neoformansis recognized and phagocytosed by macrophages. Here we investigate the role of TLR4 in the nonopsonic phagocytosis ofC. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of nonopsonized cryptococci. The increased phagocytosis observed inTlr4-/-cells was dampened by pre-treatment of macrophages with either a TLR3 inhibitor or oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated inTlr4-/-macrophages and there was a 75% decrease in phagocytosis of nonopsonized cryptococci byMsr1-/-macrophages. Furthermore, immunofluorescence imaging revealed colocalization of MSR1 and internalised cryptococci. Together, these results identify MSR1 as a key receptor for the phagocytosis of nonopsonizedC. neoformansand demonstrate TLR4/MSR1 crosstalk in the phagocytosis ofC. neoformans.
A comprehensive survey of coronaviral main protease active site diversity in 3D: Identifying and analyzing drug discovery targets in search of broad specificity inhibitors for the next coronavirus pandemic
Authors: Lubin Joseph H. , Martinusen Samantha G. , Zardecki Christine , Olivas Cassandra , Bacorn Mickayla , Balogun MaryAgnes , Slaton Ethan W. , Wu Amy Wu , Sakeer Sarah , Hudson Brian P. , Denard Carl A. , Burley Stephen K. , Khare Sagar D.
Published: January 2023

Although the rapid development of therapeutic responses to combat SARS-CoV-2 represents a great human achievement, it also demonstrates untapped potential for advanced pandemic preparedness. Cross-species efficacy against multiple human coronaviruses by the main protease (MPro) inhibitor nirmatrelvir raises the question of its breadth of inhibition and our preparedness against future coronaviral threats. Herein, we describe sequence and structural analyses of 346 unique MPro enzymes from all coronaviruses represented in the NCBI Virus database. Cognate substrates of these representative proteases were inferred from their polyprotein sequences. We clustered MPro sequences based on sequence identity and AlphaFold2-predicted structures, showing approximate correspondence with known viral subspecies. Predicted structures of five representative MPros bound to their inferred cognate substrates showed high conservation in protease:substrate interaction modes, with some notable differences. Yeast-based proteolysis assays of the five representatives were able to confirm activity of three on inferred cognate substrates, and demonstrated that of the three, only one was effectively inhibited by nirmatrelvir. Our findings suggest that comprehensive preparedness against future potential coronaviral threats will require continued inhibitor development. Our methods may be applied to candidate coronaviral MPro inhibitors to evaluate in advance the breadth of their inhibition and identify target coronaviruses potentially meriting advanced development of alternative countermeasures.
A common tolerance mechanism of bacterial biofilms to antibiotics
Authors: Zhang Yuzhen , Cai Yumin , Wang Tianmin , Wu Qile
Published: January 2023

A deeper mechanistic understanding of antibiotics tolerance exhibited by bacterial biofilms is crucial for clinical treatment and drug development. Here, we found a common binary distribution for various antibiotics in biofilms, which was determined by glucose distribution. Antibiotic penetration would be accompanied by glucose leakage. Surprisingly, the biofilm periphery exhibited constant glucose consumption and antibiotics patterns after drug treatment, while planktonic bacteria did not. Spatial multi-omics revealed that activation of lipid metabolism was responsible for continuous glucose consumption, which was channeled to thicken the cell membrane and subsequently weakened antibiotic penetration, leading to a low drug concentration in the biofilm interior - a common mechanism underlying biofilm drug tolerance. We further revealed that the biofilm-specific activation of lipid metabolism was derived from slower drug penetration and longer response time of biofilms to antibiotics, owing to lower energy metabolism and membrane potential in biofilms compared to planktonic bacteria.
An optimized workflow for analyzing extracellular vesicles as biomarkers in liver diseases
Authors: Paluschinski Martha , Loosen Sven , Kordes Claus , Keitel Verena , Kuebart Anne , Brandenburger Timo , Schöler David , Wammers Marianne , Neumann Ulf P , Luedde Tom , Castoldi Mirco
Published: January 2023

Background & Aims

Extracellular vesicles (EVs) play an important role in intercellular communication, serving as vehicles for the exchange of biological materials and being involved in the regulation of physiological processes. EVs and their associated cargoes are considered a promising source of disease-associated biomarkers. The purpose of this study was to establish an easy-to-use, reproducible, and scalable workflow to efficiently analyze EVs in the context of liver disease.

Methods

An optimized workflow was established for the pre-analytical processing and isolation of EVs from plasma and serum. Nanoparticle Tracking Analysis (NTA) was used to characterize circulating EVs in the serum of patients with nonalcoholic fatty liver disease (NAFLD), autoimmune liver disease (AIH), and animal models with impaired liver function. EVs were separated from soluble proteins by an optimized, polyethylene glycol (PEG)-based enrichment protocol. Enriched EVs were either labeled and functionally characterized by monitoring cellular uptake or lysed for biomarker identification.

Results

Circulating EVs in the serum of patients with NAFLD or AIH and in different animal models have been characterized by NTA. Here we show that both the quantity and size of EVs in the serum of patients/animal models are significantly different from those of healthy individuals. We show that isolated EVs are functional, and their uptake by acceptor cells can be quantified after fluorescence labelling. Enriched EVs were directly used to analyze RNA biomarkers. Several microRNAs, including miR-15b, -16, -21, -122 and -223, were found to be significantly up-regulated in EVs isolated from the sera of patients with NAFLD and AIH. We show that EVs transport cytokines, and that IL-2, IL-6 and IL-8 were significantly up-regulated in EVs enriched from patients with cholangiocarcinoma (CCA) compared to healthy controls.

Conclusions

The workflow presented here represents an accessible and easy-to-use approach that enables the analysis and enrichment of EVs from complex biological fluids and their preparation for functional characterization or downstream analysis. In this study, the levels of several miRNAs were found to be significantly increased in EVs isolated from AIH and NAFLD patients compared with healthy controls.

Highlights

EVs circulating in crude serum reflect the diseased stage of the donors.

Enrichment of EVs with the approach presented here efficiently separates soluble proteins from EVs, providing optimal material for further characterization.

Exosomal markers are present in the EVs-enriched fraction.

Enriched EVs are intact and are functionally taken up by acceptor cells.

Enriched EVs are suitable, and have been used for, biomarkers identification both at RNA and protein level.
Homeostatic control of an iron repressor in a GI tract resident
Authors: Wang Yuanyuan , Mao Yinhe , Chen Xiaoqing , Yang Kaiyan , Huang Xinhua , Tian Lixing , Jiang Tong , Zou Yun , Ma Xiaoyuan , Xu Chaoyue , Zhou Zili , Wu Xianwei , Pan Lei , Liang Huaping , Chen Changbin
Published: January 2023

The transition metal iron plays a crucial role in living cells. However, high level of iron is potentially toxic through the production of reactive oxygen species (ROS), serving as a deterrent to the commensal fungusCandida albicansfor colonization in the iron-rich gastrointestinal (GI) tract. We observe that the mutant lacking an iron-responsive transcription factor Hap43 is hyper-fit for colonization in murine gut. We demonstrate that high iron specifically triggers multiple post-translational modifications (PTMs) and proteasomal degradation of Hap43, a vital process guaranteeing the precision of intestinal ROS detoxification. Reduced levels of Hap43 lead to de-repression of antioxidant genes and therefore alleviate the deleterious ROS derived from iron metabolism. Our data reveal that Hap43 functions as a negative regulator for oxidative stress-adaptation ofC. albicansto gut colonization and thereby provide a new insight into understanding the interplay between iron homeostasis and fungal commensalism.

Importance

Iron homeostasis is critical for creatures.Candida albicansis one of the major commensals in the GI tract where is iron-replete environment. Transcriptional factor Hap43 was believed to repress iron utilizations genes in iron-depleted conditions for decades. However, the mystery in iron-replete conditions of Hap43 has never been uncovered. We discovered that reduced levels of Hap43 via phosphorylation-dependent nuclear export, followed by proteosome-mediated protein degradation, leads to de-repression of downstream antioxidant genes and promote its colonization in GI tract. We propose thatC. albicanshas a strict detoxification process to ensure its survival, which has important implications for understanding how the fungi survives in the mammalian host.
Co-evolutionary patterns shown inOstreococcus-virus system from the Western Baltic Sea in freshly isolated hosts and viruses
Authors: Listmann Luisa , Peters Carina , Rahlff Janina , Esser Sarah P. , Schaum Elisa
Published: January 2023

Marine viruses are a major driver of phytoplankton mortality and thereby influence biogeochemical cycling of carbon and other nutrients. In recent years, an understanding of the potential importance of phytoplankton-targeting viruses on ecosystem dynamics has emerged, but experimental investigations of host-virus interactions on a broad spatial and temporal scale are still missing. Here, we investigated in detail a phytoplankton host’s responses reacting to infections by species-specific viruses from i) distinct geographical regions and ii) different sampling seasons. Specifically, we used two species of picophytoplankton (1 µm)Ostreococcus tauriandO. mediterraneusand their viruses (size ca. 100 nm), which represent systems well-known in marine biology, but almost entirely ignored in evolutionary biology. The strains stem from different regions of the Southwestern Baltic Sea that vary in salinity and temperature. Using an experimental cross-infection set-up, we show that in this natural system evolutionary history, and thus the timing of when hosts and their associated viruses coexisted, was the main driver of infection patterns. In addition species and strain specificity underline the present understanding of rapid host-virus co-evolution.
The virus-encoded ion channel “viroporin” activity of the agnoprotein is required for BK Polyomavirus release from infected kidney cells
Authors: Swinscoe Gemma , Prescott Emma L , Hurdiss Daniel L , Morgan Ethan L , Dobson Samuel J , Edwards Thomas , Foster Richard , Welberry Smith Matthew , Macdonald Andrew
Published: January 2023

BK polyomavirus (BKPyV) is a common opportunistic pathogen and the causative agent of several diseases in transplant patients and the immunosuppressed. Despite its importance, aspects of the virus lifecycle such as how the virus exits an infected cells, remain poorly understood. The late region of the BKPyV genome encodes an auxillery protein called agnoprotein. We and others have shown that agnoprotein is an essential factor in virus release, and the loss of agnoprotein results in an accumulation of virus particles within the nucleus of an infected cell. The functions of agnoprotein necessary for this egress phenotype are not known. Here we demonstrate that agnoprotein shows properties associated with viroporins, a group of virus-encoded membrane spanning proteins that play key roles in virus infection and release. We demonstrate that agnoprotein oligomerises and perturbs membranes in cells. The development of a novel recombinant agnoprotein expression system permitted the identification of the first small molecules targeting agnoprotein. These compounds abrogated agnoprotein viroporin activity in vitro and reduced virus release, indicating that viroporin activity contributes to the phenotype observed in agnoprotein knockout viruses. The identification of channel activity should enhance the future understanding of the physiological function of agnoprotein and could represent an important target for antiviral intervention.
Coregulation of extracellular vesicle production and fluconazole susceptibility inCryptococcus neoformans
Authors: Rizzo Juliana , Trottier Adèle , Moyrand Frédérique , Coppee Jean-Yves , Maufrais Corinne , Zimbres Ana Claudia G. , Dang Thi Tuong Vi , Alanio Alexandre , Desnos-Ollivier Marie , Mouyna Isabelle , Péhau-Arnaudet Gérard , Commere Pierre-Henri , Novault Sophie , Ene Iuliana V. , Nimrichter Leonardo , Rodrigues Marcio L. , Janbon Guilhem
Published: January 2023

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novelCryptococcus neoformansregulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover,in vitrospontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen.

IMPORTANCE

Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation but thier functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogenCryptococcus neoformans.Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated inC. neoformansclinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.
The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA double strand repair through mutual regulation of their protein levels
Authors: Mennerich Daniela , Ashok Yashwanth , Vela-Rodríguez Carlos , Hentilä Heli I. , Rall-Scharpf Melanie , Wiesmüller Lisa , Prunskaite-Hyyryläinen Renata , Lehtiö Lari , Kietzmann Thomas
Published: January 2023

The DNA damage response (DDR) relies on a complex protein network to maintain genomic integrity, yet the interplay between post-translational modifiers remains poorly understood. Here, we uncover a novel regulatory axis between the E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 at DNA double-strand breaks (DSBs). Our results reveal a sophisticated feedback mechanism in which DTX3L ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself and DTX3L. This cross-regulation fine-tunes DSB repair in multiple pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ). Strikingly, the detrimental effects of USP28 depletion on these repair pathways were rescued by concurrent DTX3L knockdown. Collectively, our work uncovers a novel layer of DDR regulation in which DTX3L and USP28’s antagonistic activities calibrate cellular responses to genotoxic stress, thus identifying promising therapeutic targets to combat diseases associated with genomic instability.

Highlights

DTX3L and USP28 physically interact and colocalize in cellular sub-compartments, with the N-terminal D1-D3 domains of DTX3L primarily mediating the interaction

DTX3L ubiquitinates USP28 for degradation, while USP28 deubiquitinates itself and DTX3L, creating a sophisticated feedback mechanism.

The DTX3L-USP28 circuit influences levels of key proteins like HIF-1α, p53, and c-MYC, suggesting broader impacts on cellular stress responses.

DTX3L and USP28 cooperatively regulate multiple DSB repair pathways, including NHEJ, HR, SSA, and MMEJ, with USP28 depletion effects rescued by DTX3L silencing.
Structure of the PfRCR complex which bridges the malaria parasite and erythrocyte during invasion
Authors: Farrell Brendan , Alam Nawsad , Hart Melissa N. , Jamwal Abhishek , Ragotte Robert J. , Walters-Morgan Hannah , Draper Simon J. , Knuepfer Ellen , Higgins Matthew K.
Published: January 2023

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The five-component PfPCRCR complex, containing PfRH5, PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies or nanobodies against each of these five conserved proteins, making them the leading blood stage malaria vaccine candidates. However, little is known about the molecular mechanism by which PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. This reveals that PfRIPR consists of an ordered multi-domain core flexibly linked to an elongated tail. We test the hypothesis that PfRH5 opens to insert into the membrane, but instead show that a rigid, disulphide-locked PfRH5 can mediate efficient erythrocyte invasion. Finally, we show that the elongated tail of PfRIPR, which is the target of growth- neutralising antibodies, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. Therefore, a modular PfRIPR is linked to the merozoite membrane through an elongated tail, while its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides novel insight into the mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.
G-quadruplexes regulate miRNA biogenesis in live zebrafish embryos
Authors: Steeman Tomás J. , Weiner Andrea M. J. , David Aldana P. , Binolfi Andrés , Calcaterra Nora B. , Armas Pablo
Published: January 2023

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesisin vivoduring zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to foldin vitroas G4. MiR-150 controls the expression ofmyb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos within vitrotranscribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels ofmybmRNA and stronger phenotypes associated withmybknock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related tomybknock-down. Overall, results suggest that the G4 formed in pre-miR-150 functionsin vivoas a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis.
A New Platform for Label-Free, Proximal Cellular Pharmacodynamic Assays: Identification of Glutaminase Inhibitors Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry
Authors: Pu Fan , Radosevich Andrew J. , Bruckner Brett G. , Fontaine David A. , Panchal Sanjay C. , Williams Jon D. , Gopalakrishnan Sujatha M. , Elsen Nathaniel L.
Published: January 2023

Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently low throughput which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the less resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis but liquid chromatography-mass spectrometry methods are low throughput and unable to fully support structure-activity relationships efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling process, and low cross contamination. Here we demonstrate the capability of IR-MALDESI for detection of diverse analytes directly from cells and report the development of a high throughput label free, proximal cellular pharmacodynamic assay using IR-MALDESI for discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a ∼100,000 compound cellular screen. Hits from the screening were confirmed by retesting in dose-response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.