bluuebunny/crossref_metadata_2025 huggingface dataset:
Xanthomonas oryzaepv.oryzaetype-III effector TAL9b targets a broadly conserved disease susceptibility locus to promote pathogenesis in rice
Authors: Gokulan C.G. , Deb Sohini , Gaur Namami , Masade Apoorva , Gattu Niranjan , Rennya P.R. , Sao Nisha , James Donald , Sonti Ramesh V. , Patel Hitendra K.
Published: May 2024

Xanthomonas oryzaepv.oryzae(Xoo), the causal agent of bacterial blight of rice, translocates multiple Transcription Activator-Like Effectors (TALEs) into rice cells. The TALEs localize to the host cell nucleus, where they bind to the DNA in a sequence-specific manner and enhance gene expression to promote disease susceptibility. Xoo strain PXO99Aencodes nineteen TALEs, but the host targets of all these TALEs have not been defined. A meta-analysis of rice transcriptome profiles revealed a gene annotated as flavonol synthase/flavanone-3 hydroxylase (henceforthOsS5H/FNS-03g) to be highly induced upon Xoo infection. Further analyses revealed that this gene is induced by PXO99Ausing TAL9b, a broadly conserved TALE of Xoo. Disruption oftal9brendered PXO99Aless virulent. OsS5H/FNS-03g functionally complemented itsArabidopsishomologue AtDMR6, a well-studied disease susceptibility locus. Biochemical analyses suggested that OsS5H/FNS-03g is a bifunctional protein with Salicylic Acid 5’ Hydroxylase (S5H) and Flavone Synthase-I (FNS-I) activities. Further, an exogenous application of apigenin on rice leaves, the flavone that is enzymatically produced by OsS5H/FNS-03g, promoted virulence of PXO99Atal9b-. Overall, our study suggests that OsS5H/FNS-03g is a bifunctional enzyme and its product apigenin is potentially involved in promoting Xoo virulence.
Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in 40S small ribosomal subunit
Authors: Querido Jailson Brito , Sokabe Masaaki , Díaz-López Irene , Gordiyenko Yuliya , Zuber Philipp , Du Yifei , Albacete-Albacete Lucas , Ramakrishnan V. , Fraser Christopher S.
Published: May 2024

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding andin vitrotranslation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5′ UTR of mRNA.
A novel induced pluripotent stem cell model of Schwann cell differentiation revealsNF2- related gene regulatory networks of the extracellular matrix
Authors: Lazaro Olivia , Li Sihong , Carter William , Awosika Oluwamayowa , Robertson Sylvia , Hickey Brooke E. , Angus Steven P. , House Austin , Clapp Wade D. , Qadir Abdul S. , Johnson Travis S. , Rhodes Steven D.
Published: May 2024

Schwann cells are vital to development and maintenance of the peripheral nervous system and their dysfunction has been implicated in a range of neurological and neoplastic disorders, includingNF2-related schwannomatosis. We developed a novel human induced pluripotent stem cell (hiPSC) model to study Schwann cell differentiation in health and disease. We performed transcriptomic, immunofluorescence, and morphological analysis of hiPSC derived Schwann cell precursors (SPCs) and terminally differentiated Schwann cells (SCs) representing distinct stages of development. To validate our findings, we performed integrated, cross-species analyses across multiple external datasets at bulk and single cell resolution. Our hiPSC model of Schwann cell development shared overlapping gene expression signatures with human amniotic mesenchymal stem cell (hAMSCs) derived SCs andin vivomouse models, but also revealed unique features that may reflect species-specific aspects of Schwann cell biology. Moreover, we identified gene co-expression modules that are dynamically regulated during hiPSC to SC differentiation associated with ear and neural development, cell fate determination, theNF2gene, and extracellular matrix (ECM) organization. By cross-referencing results between multiple datasets, we identified new genes potentially associated withNF2expression. Our hiPSC model further provides a tractable platform for studying Schwann cell development in the context of human disease.
CetZ1-dependent polar assembly of the motility machinery in haloarchaea
Authors: Brown Hannah J. , Islam Md Imtiazul , Ruan Juanfang , Baker Matthew A. B. , Ithurbide Solenne , Duggin Iain G.
Published: May 2024

Archaeal CetZ cytoskeletal proteins are a major group of the tubulin superfamily involved in the development of motile rod-shaped cells. In the model archaeonHaloferax volcanii, it has been unknown whether CetZs contribute to motility solely through the effects of rod cell shape on swimming speed, or in other ways too. Here, we used cryo-electron and fluorescence microscopy to observe cell surface filaments and the assembly of the motility machinery in cells carryingcetZ1orcetZ2deletion, overexpression, or dominant GTPase-deficient mutations. CetZ1 was found to play important roles in the polar assembly of motility marker proteins, including ArlD1, a constituent of the archaellum base, the chemotaxis sensory array adapter CheW1, and signal transducer CheY. Notably, overexpression ofcetZ1reduced motility and the assembly of the motility machinery markers but did not affect the rod shape of motile cells. Dual protein localization studies further suggested that CetZ1 is not a direct stoichiometric component of the motility machineries but promotes their assembly via a significant role in organisation or structure of the cell poles.

Significance

This study demonstrates that cytoskeletal proteins (CetZs) can contribute to assembly of the motility machinery in archaea. In the model organism,H. volcanii, this function is expected to contribute significantly to the overall impact of CetZ1 on swimming motility, in addition to its established role in rod cell formation generating a streamlined shape. Our study also provides the first evidence that archaeal CetZ tubulin superfamily proteins can have structural or organisational roles for the assembly of protein complexes on the cell surface and cell poles. The multiple functions and subcellular locations of CetZ1 during motile cell development are reminiscent of eukaryotic cytoskeletal proteins and the roles of tubulin in basal bodies during the development of the eukaryotic flagellum.
What does your gut tell you? Influences of a zombie-making and generalist fungal entomopathogen on carpenter ant micro- and mycobiota
Authors: Vermeulen Sophia , Forsman Anna M , de Bekker Charissa
Published: May 2024

Microbiome composition impacts many host aspects including health, nutrition, reproduction, and behavior. This warrants the recent uptick in insect microbiota research across species and ecosystems. Commensurate with this, the bacterial microbiome of the ant Camponotus floridanus has been well characterized across body regions and maturation levels. However, potential effects of entomopathogens on the gut microbiome, and the fungal communities therein, are yet to be assessed. Investigation of the microbiome during infection could provide insight into entomopathogenic infection and manipulation strategies and inform effective biopesticide strategies. Additionally, the mycobiome remains often overlooked despite playing a vital role in gut ecology with potential implications for health and infection outcomes. To improve our limited understanding of fungal infections in insects, and ants in particular, we characterized the effects of two entomopathogens with different infection strategies on the gut micro- and mycobiota of C. floridanus over time; Ophiocordyceps camponoti-floridani and Beauveria bassiana. Specialist, zombie-making O. camponoti-floridani fungi hijack the behavior of C. floridanus ants over three weeks, causing them to find an elevated position, and fix themselves in place with their mandibles. This summiting behavior is adaptive to Ophiocordyceps as the ant transports the fungus to conditions that favor fruiting body development, spore production, dispersal, and transmission. In contrast, the generalist entomopathogen B. bassiana infects and kills the ant within a few days, without the induction of obvious fungus-adaptive behaviors. By comparing healthy ants with Beauveria- and Ophiocordyceps-infected ants we aimed to 1) describe the dynamics of the micro- and mycobiome of C. floridanus during infection, and 2) determine if the effects on gut microbiota are distinctive between fungi that have different infection strategies. While Beauveria did not measurably affect the ant host micro-and mycobiome, Ophiocordyceps did, especially for the mycobiome. Moreover, ants that were sampled during Ophiocordyceps-adaptive summiting behavior had a significantly different micro- and mycobiome composition compared to healthy controls and those sampled before and after manipulation took place. This suggests that the host microbiome might have a role to play in the manipulation strategy of Ophiocordyceps.
Pore engineering as a general strategy to improve protein-based enzyme nanoreactor performance
Authors: Kwon Seokmu , Andreas Michael P. , Giessen Tobias W.
Published: May 2024

Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and co-localization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins – microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for future biotechnological and biomedical applications.
Perceptual consequences of retinal stabilization with a high-frequency LCD display
Authors: Li Yuanhao H. , Rucci Michele
Published: May 2024

Several recent studies have shown decreased sensitivity when stimuli are immobilized on the retina, a procedure known as retinal stabilization. Because of the technical challenges inherent in this procedure, studies have either used fast-phosphor CRT displays or directly updated the stimulus on the retina by means of adaptive optics scanning laser ophthalmoscopes. Both display systems provide brief pulses to the retina, raising the question of whether this flicker contributed to perceptual effects. Here we report the results of retinal stabilization experiments conducted with LCD monitors at high refresh rates. Results replicate previous findings of high-frequency impairments under retinal stabilization. These data provide further support for a functional role of small eye movements during visual fixation.
IL-2 enhances effector function but suppresses follicular localization of CD8+T cells in chronic infection
Authors: Chen Yaping , Zhou Pengcheng , Gubser Patrick Marc , Leong Yew Ann , He Jing , Wei Yunbo , Makota Fadzai Victor , Pazhouhandeh Mehrdad , Zheng Ting , Yunis Joseph , Li Zhanguo , Kallies Axel , Yu Di
Published: May 2024

Cytotoxic CD8+T cells, essential in combating viral infections and cancer, become dysfunctional from prolonged antigen exposure. Precursors of exhausted T (TPEX) cells are pivotal in sustaining immune responses in chronic diseases and mediating immunotherapy efficacy. They also control viral infection within B-cell follicles, facilitated by CXCR5 expression. How cytokines regulate TPEXcell fate and follicular entry is not well understood. We reveal that IL-2 treatment enhances CD8+T cell effector functions in chronic LCMV infection but hinders CXCR5+TPEXcell formation and infection control within B-cell follicles. Mechanistically, IL-2 suppresses TPEXcell differentiation in a STAT5 and BLIMP1-dependent manner. Using an IL-2 fusion protein targeting CD122, we shifted the differentiation towards CX3CR1+T cells with increased effector function. Clinical observations with low-dose IL-2 in autoimmune disease confirmed IL-2’s inhibitory effect on CXCR5+TPEXcells, underscoring IL-2’s crucial regulatory role and therapeutic potential in modulating TPEXand effector T cell generation.
Pitfalls and recommendations for large-scale SNP genotyping in a non-model endangered species: the southern damselflies (Coenagrion mercuriale) as a case study
Authors: Lévêque Agathe , Arnaud Jean-François , Vignon Vincent , Mazoyer Clément , Godé Cécile , Duputié Anne
Published: May 2024

Genomic markers are essential tools for studying species of conservation concern, yet non-model species often lack a genome reference. Here we describe a methodology for identifying and genotyping thousands of SNP loci in the southern damselfly (Coenagrion mercuriale), a bioindicator of freshwater stream quality classified as near-threatened, with locally declining populations. We used a hybrid approach combining reduced representation sequencing and target enrichment. First, we identified putative SNP loci using ddRADseq andde novoassembly. Then, single primer enrichment technology targeted 6,000 of these SNPs across 1,920 individuals. Challenges encountered included sequence recapture failure, coverage depth discrepancies, and aberrantFISvalues. We provide recommendations to address such issues. After multiple filtering, we retained 2,092 SNPs. We used them to characterise rear-edge populations of the southern damselfly in Northern France, a region where populations are sparsely distributed. Previous surveys utilising microsatellite markers allowed comparison of genetic diversity and differentiation estimates. Consistent with prior findings, genetic diversity estimates were similar across the studied populations that showed no sign of inbreeding. SNP markers exhibited greater resolution in detecting fine-scaled genetic structure, identifying two putative hybrids in adjacent populations, a feat unattainable with microsatellite loci. Altogether, this study highlighted the ongoing challenge of large-scale SNP genotyping using target sequencing techniques in non-model species to set conservation guidelines. Nonetheless, these new markers showed greater statistical power in identifying conservation units and offered the promise of greater precision in the identification of admixture events or the estimation of key population parameters such as effective population size.
Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM
Authors: Li Weicheng , Yang Haoyun , Stachowski Kye , Norris Andrew S , Lichtenthal Katie , Kelly Skyler , Gollnick Paul , Wysocki Vicki H , Foster Mark P
Published: May 2024

Homotropic cooperativity is widespread in biological regulation. The homo-oligomeric ring-shaped trp RNA binding attenuation protein (TRAP) from bacillus binds multiple tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the trp operon mRNA. Ligand-activated binding to this specific RNA sequence regulates downstream biosynthesis of Trp in a feedback loop. Characterized TRAP variants form 11- or 12-mer rings and bind Trp at the interface between adjacent subunits. Various studies have shown that a pair of loops that gate each Trp binding site is flexible in the absence of the ligand and become ordered upon ligand binding. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity we engineered dodecameric (12-mer) TRAP variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated one of the protomers such that only every other site was competent for Trp binding. Thermodynamic and structural studies using native mass spectrometry, NMR spectroscopy, and cryo-EM provided unprecedented detail into the thermodynamic and structural basis for the observed ligand binding cooperativity. Such insights can be useful for understanding allosteric control networks and for the development of new ones with defined ligand sensitivity and regulatory control.
Spontaneous HFO Sequences Reveal Propagation Pathways for Precise Delineation of Epileptogenic Networks
Authors: Cai Zhengxiang , Jiang Xiyuan , Bagić Anto , Worrell Gregory A. , Richardson Mark , He Bin
Published: May 2024

Abstract

Epilepsy, a neurological disorder affecting millions worldwide, poses great challenges in precisely delineating the epileptogenic zone – the brain region generating seizures – for effective treatment. High-frequency oscillations (HFOs) are emerging as promising biomarkers; however, the clinical utility is hindered by the difficulties in distinguishing pathological HFOs from non- epileptiform activities at single electrode and single patient resolution and understanding their dynamic role in epileptic networks. Here, we introduce an HFO-sequencing approach to analyze spontaneous HFOs traversing cortical regions in 40 drug-resistant epilepsy patients. This data- driven method automatically detected over 8.9 million HFOs, pinpointing pathological HFO- networks, and unveiled intricate millisecond-scale spatiotemporal dynamics, stability, and functional connectivity of HFOs in prolonged intracranial EEG recordings. These HFO sequences demonstrated a significant improvement in localization of epileptic tissue, with an 818.47% increase in concordance with seizure-onset zone (mean error: 2.92 mm), compared to conventional benchmarks. They also accurately predicted seizure outcomes for 90% AUC based on pre-surgical information using generalized linear models. Importantly, this mapping remained reliable even with short recordings (mean standard deviation: 3.23 mm for 30-minute segments). Furthermore, HFO sequences exhibited distinct yet highly repetitive spatiotemporal patterns, characterized by pronounced synchrony and predominant inward information flow from periphery towards areas involved in propagation, suggesting a crucial role for excitation-inhibition balance in HFO initiation and progression. Together, these findings shed light on the intricate organization of epileptic network and highlight the potential of HFO-sequencing as a translational tool for improved diagnosis, surgical targeting, and ultimately, better outcomes for vulnerable patients with drug-resistant epilepsy.

One Sentence Summary

Pathological fast brain oscillations travel like traffic along varied routes, outlining recurrently visited neural sites emerging as critical hotspots in epilepsy network.
Optimized AAV capsids for diseases of the basal ganglia show robust potency and distribution in adult nonhuman primates
Authors: Leib DE , Chen YH , Tecedor L , Ranum PT , Keiser MS , Lewandowski BC , Carrell EM , Arora S , Huerta-Ocampo I , Liu X , Davidson BL
Published: May 2024

Huntington’s disease and other disorders of the basal ganglia create challenges for biomolecule-based medicines given the poor accessibility of these deep brain structures following intracerebral or intravascular delivery. Additionally, for adeno-associated viruses (AAVs) intravascular delivery exposes peripheral tissues to the vast majority of the therapy, increasing the risk of immune responses and the quantity and associated cost of goods required for therapeutically relevant brain penetration levels. Here, we found that low dose, low volume delivery of unbiased AAV libraries into a focused brain region allowed recovery of novel capsids capable of broad access to key deep brain and cortical structures relevant for human therapies at doses orders of magnitude lower than used in current clinical trials. One such capsid, AAV-DB-3, provided transduction of up to 45% of medium spiny neurons in the adult NHP striatum, along with substantial transduction of relevant deep layer neurons in the cortex. Notably, AAV-DB-3 behaved similarly in mice as in NHPs and also potently transduced human neurons derived from induced pluripotent stem cells. Thus, AAV-DB-3 provides a unique AAV for network level brain gene therapies that translates up and down the evolutionary scale for preclinical studies and eventual clinical use.
Individual variability in the relationship between physiological and resting-state fMRI metrics
Authors: Moia Stefano , Chen Gang , Uruñuela Eneko , Stickland Rachael C. , Termenon Maite , Caballero-Gaudes César , Bright Molly G.
Published: May 2024

Cerebrovascular Reactivity (CVR), the brain’s vascular response to a vasodilatory stimulus, can be measured using fMRI during breathing challenges that modulate arterial CO2levels. CVR is an important indicator of cerebrovascular health, although its estimation can be challenging due to the extra experimental setup and/or the subject compliance required. To overcome these limitations, summary metrics based on resting state fluctuations (RSF), such as the (fractional) amplitude of low frequency fluctuations (f/ALFF) and the resting state fluctuation amplitude (RSFA), have been proposed as alternative estimates of CVR, as they are frequently associated with vascular and physiological factors. Previous studies have reported a significant relationship between CVR estimates obtained by means of respiratory paradigms and RSF metrics. However, the total sample sizes, considering both subjects and sessions, are typically small, and not all studies agree on the degree of the relationship. Furthermore, to our knowledge, these studies have only reported cross-sectional analyses, whereas intra-subject longitudinal relationships between CVR estimates and RSF metrics are unknown. Leveraging a unique dense sampling dataset in which resting state and breath-hold multi-echo fMRI were collected in 7 subjects with 10 sessions each, we provide evidence of high individual variability in the inter-session (i.e. intra-subject) relationship between RSF metrics and CVR. These results indicate that RSF metrics might not be a suitable proxy of CVR in clinical settings or for BOLD signal calibration as they may not properly account for intra-individual physiological variations in BOLD fMRI data.
Vestibular Modulation by Stimulant Derivatives in a Pentameric Ligand-Gated Ion Channel
Authors: Karlsson Emelia , Andén Olivia , Fan Chen , Fourati Zaineb , Haouz Ahmed , Zhuang Yuxuan , Howard Rebecca J , Delarue Marc , Lindahl Erik
Published: May 2024

Allosteric modulation of pentameric ligand-gated ion channels (pLGICs) is critical to the action of neurotransmitters and many psychoactive drugs. However, details of their modulatory mechanisms remain unclear, especially beyond the orthosteric neurotransmitter-binding sites. The recently reported prokaryotic channel sTeLIC, a pH-gated homolog of eukaryotic receptors in the pLGIC family, is thought to be modulated by aromatic compounds via a relatively uncharacterized modulatory site in the extracellular vestibule. Here, we show that sTeLIC is sensitive to potentiation by psychostimulant derivatives. By determining new cryo-EM and X-ray structures in closed and open states, and testing the impact of targeted mutations on electrophysiological behavior, we show that several amphiphilic compounds preferentially bind a vestibular pocket in the contracted open-state extracellular domain. This work provides a detailed structure-function mechanism for allosteric potentiation via a noncanonical lig- and site, with potential conservation in eukaryotic pentameric ligand-gated ion channels.
Perinatal maternal undernutrition in baboons modulates hepatic mitochondrial function but not metabolites in aging offspring
Authors: Adekunbi Daniel A. , Yang Bowen , Huber Hillary F. , Riojas Angelica M. , Moody Alexander J. , Li Cun , Olivier Michael , Nathanielsz Peter W. , Clarke Geoffery D. , Cox Laura A. , Salmon Adam B.
Published: May 2024

We previously demonstrated in baboons that maternal undernutrition (MUN), achieved by 70 % of control nutrition, impairs fetal liver function, but long-term changes associated with aging in this model remain unexplored. Here, we assessed clinical phenotypes of liver function, mitochondrial bioenergetics, and protein abundance in adult male and female baboons exposed to MUN during pregnancy and lactation and their control counterparts. Plasma liver enzymes were assessed enzymatically. Liver glycogen, choline, and lipid concentrations were quantified by magnetic resonance spectroscopy. Mitochondrial respiration in primary hepatocytes under standard culture conditions and in response to metabolic (1 mM glucose) and oxidative (100 µM H2O2) stress were assessed with Seahorse XFe96. Hepatocyte mitochondrial membrane potential (MMP) and protein abundance were determined by tetramethylrhodamine ethyl ester staining and immunoblotting, respectively. Liver enzymes and metabolite concentrations were largely unaffected by MUN, except for higher aspartate aminotransferase levels in MUN offspring when male and female data were combined. Oxygen consumption rate, extracellular acidification rate, and MMP were significantly higher in male MUN offspring relative to control animals under standard culture. However, in females, cellular respiration was similar in control and MUN offspring. In response to low glucose challenge, only control male hepatocytes were resistant to low glucose-stimulated increase in basal and ATP-linked respiration. H2O2did not affect hepatocyte mitochondrial respiration. Protein markers of mitochondrial respiratory chain subunits, biogenesis, dynamics, and antioxidant enzymes were unchanged. Male-specific increases in mitochondrial bioenergetics in MUN offspring may be associated with increased energy demand in these animals. The similarity in systemic liver parameters suggests that changes in hepatocyte bioenergetics capacity precede detectable circulatory hepatic defects in MUN offspring and that the mitochondria may be an orchestrator of liver programming outcome.
In vitro heterochronic parabiosis identifies pigment epithelium-derived factor as a systemic mediator of rejuvenation by young blood
Authors: Wang Xizhe , Tazearslan Cagdas , Kim Seungsoo , Guo Qinghua , Contreras Daniela , Yang Jiping , Hudgins Adam D. , Suh Yousin
Published: May 2024

Several decades of heterochronic parabiosis (HCPB) studies have demonstrated the restorative impact of young blood, and deleterious influence of aged blood, on physiological function and homeostasis across tissues, although few of the factors responsible for these observations have been identified. Here we develop an in vitro HCPB system to identify these circulating factors, using replicative lifespan (RLS) of primary human fibroblasts as an endpoint of cellular health. We find that RLS is inversely correlated with serum donor age and sensitive to the presence or absence of specific serum components. Through in vitro HCPB, we identify the secreted protein pigment epithelium-derived factor (PEDF) as a circulating factor that extends RLS of primary human fibroblasts and declines with age in mammals. Systemic administration of PEDF to aged mice reverses age-related functional decline and pathology across several tissues, improving cognitive function and reducing hepatic fibrosis and renal lipid accumulation. Together, our data supports PEDF as a systemic mediator of the effect of young blood on organismal health and homeostasis and establishes our in vitro HCPB system as a valuable screening platform for the identification of candidate circulating factors involved in aging and rejuvenation.
QuAPPro: An R/shiny app for Quantification and Alignment of Polysome Profiles
Authors: Schiller Chiara , Reitter Sonja , Lehmann Janina A. , Fenzl Kai , Schott Johanna
Published: May 2024

Polysome profiling is a powerful technique to study mRNA translation. After separation of ribosomal subunits from monosomes and polysomes by ultracentrifugation on sucrose density gradients, a UV absorbance profile is recorded during elution. This profile can be used to assess global translational activity, or reveal changes in ribosome biogenesis or translation elongation. In parallel to UV absorbance profiles, it is also possible to record fluorescence to measure the association of fluorescently tagged proteins with ribosomes or polysomes. To this end, the area under subsections of the UV/fluorescence profiles needs to be quantified carefully. In addition, alignment of profiles in one graph helps to visualize differences. With QuAPPro, we present the first interactive web app that allows quantification and alignment of polysome profiles, independently of the device or software that was used to generate the profiles. This user-friendly tool does not only speed up the analysis of polysome profiles but also facilitates reproducibility and documentation of the process.
High-Resolution Epitope Mapping for Glycosylated Antigens: Pembrolizumab-PD1 as a Case Study
Authors: Zhang Sara , Simmons Calvin , Young Mike , Pan Jason
Published: May 2024

High-resolution epitope mapping is important for in-depth activity assessment of antibodies and understanding of their functions, however, glycosylated antigens are challenging for many methods including crystallography. PD1 is a highly glycosylated antigen, and with the traditional HDX-MS method, only 51% sequence coverage could be obtained with multiple epitope residues undetected for Pembrolizumab (Keytruda). By implementing glyco-peptide detection, subzero temperature LC-MS and electron based MSMS fragmentation, the FineMapping methodology enabled 100% sequence coverage and complete epitope characterization for the Pembrolizumab-PD1 system, with amino acid level resolution. As 50% to 70% of proteins in the cell are glycosylated, the middle-down HDX-MS based FineMapping technology presented here should find its wide application on these protein targets. Also, FineMapping detects antibody epitopes directly in solution, without any mutation or modification to either the antigen or the antibody. And its amino acid level resolution combined with its low cost, minimal sample consumption, fast turnaround time, and no need of mutant library or crystallization makes it a competitive methodology for high-resolution epitope mapping.
Alcohol use disorder and body mass index show genetic pleiotropy and shared neural associations
Authors: Malone Samantha G. , Davis Christal N. , Piserchia Zachary , Setzer Michael R. , Toikumo Sylvanus , Zhou Hang , Winterlind Emma L. , Gelernter Joel , Justice Amy , Leggio Lorenzo , Rentsch Christopher T. , Kranzler Henry R. , Gray Joshua C.
Published: May 2024

Despite neurobiological overlap, alcohol use disorder (AUD) and body mass index (BMI) show minimal genetic correlation (rg), possibly due to mixed directions of shared variants. We applied MiXeR to investigate shared genetic architecture between AUD and BMI, conjunctional false discovery rate (conjFDR) to detect shared loci and their directional effect, Local Analysis of (co)Variant Association (LAVA) for local rg, Functional Mapping and Annotation (FUMA) to identify lead single nucleotide polymorphisms (SNPs), Genotype-Tissue Expression (GTEx) to examine tissue enrichment, and BrainXcan to assess associations with brain phenotypes. MiXeR indicated 82.2% polygenic overlap, despite a rgof −.03. ConjFDR identified 132 shared lead SNPs, with 53 novel, showing both concordant and discordant effects. GTEx analyses identified overexpression in multiple brain regions. Amygdala and caudate nucleus volumes were associated with AUD and BMI. Opposing variant effects explain the minimal rg between AUD and BMI, with implicated brain regions involved in executive function and reward, clarifying their polygenic overlap and neurobiological mechanisms.
Molecular identification of whole squids and calamari at fairs and markets in regions of Latin America
Authors: Paiva Bianca Lima , Souza Rodrigues Alan Erik , de Freitas Almeida Igor Oliveira , Haimovici Manuel , Markaida Unai , Charvet Patricia , Faria Vicente Vieira , Batista Bruno B. , Gomes Tomás Acácio Ribeiro , da Silva Rodrigues-Filho Luis Fernando , Ready Jonathan Stuart , de Luna Sales João Bráullio
Published: May 2024

In recent decades, the commercial importance of cephalopods has increased considerably, being an important fishing resource around the world. However, during the preparation for commercialization of those species, especially those sold in markets, they suffer the process known as“calamari”which includes removing and separating the head, arm, skin or even having the body structure cut into rings, which ends up making it difficult or often prevents the identification of the species, which can lead to replacements. In this sense, the present study aimed to use the large ribosomal region, rrnL (also known as 16S rDNA) to genetically identify cephalopod species sold in markets and fairs in Latin America. Whole and processed samples were collected from supermarkets and directly from local fishers the approximate collection location. Each generated sequence was submitted to the websiteGenbankfor molecular comparison and included in the database for subsequent genetic identification. Comparison of sequences using theBlastnrevealed the presence of eight species that are widely traded in the Latin American region. Our results indicate labeling errors in samples from the State of Pará that contained the speciesDosidicus gigas(d’ Orbigny, 1823) found only in the Pacific Ocean and were generically labeled as “National Lula”. No type of substitution was found among the samples that were being sold at fairs and markets, only labeling errors. Thus, our results demonstrate the effectiveness of the rrnL for identifying species and evaluating labeling errors.
Eco-evolutionary dynamics of anthelmintic resistance in soil-transmitted helminths
Authors: Patel Swati , Lyberger Kelsey , Vegvari Carolin , Gulbudak Hayriye
Published: May 2024

Anthelmintic resistance (AR) of helminth parasites against the most widely available drugs is an ongoing concern for both human and livestock-infecting species. Indeed, there has been substantial evidence of AR in livestock but less in humans, which may be due to a variety of reasons. In this paper, we develop an eco-evolutionary model that couples the life cycle of these parasites with their underlying evolution in a single biallelic genetic locus that confers resistance to treatment drugs. We determine the critical treatment frequency needed to effectively eliminate the population, for a fixed drug efficacy (without evolution) and use this to classify three qualitative distinct behaviors of the eco-evolutionary model. Then, we describe how aspects of the life cycle influence which qualitative outcome is achieved and the spread of the resistance allele, comparing across parameterized models of human- and livestock-infecting species. For all but one species, we find that lower fecundity rates and lower contact rates speed the spread of resistance, while lower larval death slows it down. The life cycle parameters ofAncylostoma duodenaleandOstertagia circumcinctaare associated with the fastest and slowest spread of resistance, respectively. We discuss the mechanistic reason for these results.
Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration
Authors: Moreno Sofia Ines Garcia , Limani Fabian , Ludwig Iina , Gilbert Catherine , Pifl Christian , Hnasko Thomas S. , Steinkellner Thomas
Published: May 2024

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson’s disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigrapars compacta(SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any DA neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute DA neurodegeneration.
Insect abundance and richness response to ecological reclamation on well pads 5-12 years into succession in a semi-arid natural gas field
Authors: Curran Michael F. , Allison Jasmine , Robinson Timothy J. , Robertson Blair L. , Knudson Alexander H. , Bott Bee M.M. , Bower Steven , Saleh Bobby M.
Published: May 2024

Natural gas extraction is a critical driver of the economy in western North America. Ecological reclamation is important to ensure surface disturbance impacts associated with natural gas development are not permanent and to assist native biota. Previous studies in semi-arid natural gas fields within Sublette County, Wyoming, USA have shown insects respond favorably to 1-3-year-old well pads undergoing reclamation compared to older successional, reference vegetation communities dominated by Wyoming big sagebrush (Artemisia tridentaspp.Wyomingensis). Here we examined well pads which were initially seeded between 5, 8, 10, 11, and 12 prior to our study. We used a free, image-based software called SamplePoint to quantify vegetation on these well pads and adjacent reference areas from high-resolution cell phone camera pictures. Insects were collected with a sweep net and identified to the family and morphospecies level. Statistical analyses were conducted to compare both vegetation and insect communities between reclamation sites and their paired reference area. We found little statistical difference between vegetation communities across our study, but found significantly more insect abundance in 3 of 5 years and significantly higher family and species richness in 4 of 5 years. Our results suggest reclamation of natural gas well pads within an old successional stand of sagebrush continues to support higher levels of insect biodiversity and abundance for at least 12 years. As insects are the most diverse group of animals on Earth and because they provide a wide array of ecosystem services, our findings suggest ecological reclamation plays an important role in returning biodiversity and ecosystem functionality to a semi-arid and old successional sagebrush-steppe ecosystem.
Syngeneic natural killer cell therapy activates dendritic and T cells in metastatic lungs and effectively treats low-burden metastases
Authors: Huang Shih-Wen , Lai Yein-Gei , Liao Hao-Ting , Chang Chin-Ling , Ma Ruo-Yu , Chen Yung-Hsiang , Liou Yae-Huei , Wu Zhen-Qi , Wu Yu-Chen , Liu Ko-Jiunn , Huang Yen-Tsung , Yang Jen-Lung , Dai Ming-Shen , Liao Nan-Shih
Published: May 2024

Natural killer (NK) cells can control metastasis through cytotoxicity and IFN-γ production independently of T cells in experimental metastasis mouse models. The inverse correlation between NK activity and metastasis incidence supports a critical role for NK cells in human metastatic surveillance. However, autologous NK cell therapy has shown limited benefit in treating patients with metastatic solid tumors. Using a spontaneous metastasis mouse model of MHC-I+breast cancer, we found that transfer of IL-15/IL-12-conditioned syngeneic NK cells after primary tumor resection promoted long-term survival of mice with low metastatic burden and induced a tumor-specific protective T cell response that is essential for the therapeutic effect. Furthermore, NK cell transfer augments activation of conventional dendritic cells (cDCs), Foxp3−CD4+T cells and stem cell-like CD8+T cells in metastatic lungs, to which IFN-γ of the transferred NK cells contributes significantly. These results imply direct interactions between transferred NK cells and endogenous cDCs to enhance T cell activation. We conducted an investigator-initiated clinical trial of autologous NK cell therapy in six patients with advanced cancer and observed that the NK cell therapy was safe and showed signs of effectiveness. These findings indicate that autologous NK cell therapy is effective in treating established low burden metastases of MHC-I+tumor cells by activating the cDC-T cell axis at metastatic sites.
Incorporation of Epstein-Barr viral variation implicates significance of LMP1 in survival prediction and prognostic subgrouping in Burkitt lymphoma
Authors: Kim Isaac E. , Oduor Cliff , Stamp Julian , Luftig Micah A. , Moormann Ann M. , Crawford Lorin , Bailey Jeffrey A.
Published: May 2024

While Epstein-Barr virus (EBV) plays a role in Burkitt lymphoma (BL) tumorigenesis, it is unclear if EBV genetic variation impacts clinical outcomes. From 130 publicly available whole-genome tumor sequences of EBV-positive BL patients, we used least absolute shrinkage and selection operator (LASSO) regression and Bayesian variable selection models within a Cox proportional hazards framework to select the top EBV variants, putative driver genes, and clinical features associated with patient survival time. These features were incorporated into survival prediction and prognostic subgrouping models. Our model yielded 22 EBV variants including seven in LMP1 as most associated with patient survival time. Using the top EBV variants, driver genes, and clinical features, we defined three prognostic subgroups that demonstrated differential survival rates, laying the foundation for incorporating EBV variants such as those in LMP1 as predictive biomarker candidates in future studies.
Protein S-glutathionylation confers cell resistance to ferroptosis
Authors: Ju Yi , Zhang Yuting , Qiao Yiming , Tian Xiaolin , Zheng Yufan , Yang Tao , Niu Baolin , Li Xiaoyun , Yu Liu , Liu Zhuolin , Wu Yixuan , Zhi Yang , Dong Yinuo , Xu Qingling , Wang Xuening , Wang Xiaokai , Mao Yimin , Li Xiaobo
Published: May 2024

Ferroptosis is a type of cell death that is strongly associated with the cellular redox state. Glutathione is the key to buffering lipid peroxidation in ferroptosis and can also modify proteins by S-glutathionylation under oxidative stress. Here, we showed that the strong associations among glutathione pools, protein S-glutathionylation, and susceptibility to ferroptosis existed broadly in ferroptosis induced by erastin or acetaminophen. Deficiency of CHAC1, a glutathione-degrading enzyme, led to decreased glutathione pools and reduced protein S-glutathionylation, improved liver function and attenuated hepatocyte ferroptosis upon acetaminophen challenge, which could be retarded by CHAC1 overexpression. We conducted quantitative redox proteomics in primary mouse hepatocytes to identify glutathione pool-sensitive S-glutathionylated proteins and found that S-glutathionylation is required to maintain the function of ADP-ribosylation factor 6 (ARF6). Our data suggest that aberrant ARF6 S-glutathionylation increases the labile iron pool by delaying the recycling of transferrin receptors, thereby promoting ferroptosis. Our study reveals the importance of protein S-glutathionylation in conferring cell resistance to ferroptosis.

HIGHLIGHTS

Highly upregulated CHAC1 decreases glutathione pools and protein S-glutathionylation.

Reduced protein S-glutathionylation associated with decreased glutathione pools promotes ferroptosis.

S-glutathionylation of ARF6 at Cys90 promotes ARF6 activation.

Reduced S-glutathionylation of ARF6 provides a labile iron pool to drive ferroptosis.
Set2 and H3K36 regulate theDrosophilamale X chromosome in a context-specific manner, independent from MSL complex spreading
Authors: Salzler Harmony R. , Vandadi Vasudha , Matera A. Gregory
Published: May 2024

Dosage compensation inDrosophilainvolves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions of MSL3 (male-specific lethal 3) with histone marks; in particular, Set2- dependent H3 lysine-36 trimethylation (H3K36me3). However, Set2 might affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Further, it is difficult to parse male-specific effects from those that are simply X- specific. To discriminate among these possibilities, we employed genomic approaches inH3K36(residue) andSet2(writer) mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global male-specific reduction of X-genes inSet2/H3K36mutants, the effects were heterogeneous. We identified cohorts of genes whose expression was significantly altered following loss of H3K36 or Set2, but the changes were in opposite directions, suggesting that H3K36me states have reciprocal functions. In contrast toH4K16Rcontrols, analysis of combinedH3.2K36R/H3.3K36Rmutants neither showed consistent reduction in X-gene expression, nor any correlation with MSL3 binding. Examination of other developmental stages/tissues revealed additional layers of context-dependence. Our studies implicate BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 directly recruits the MSL complex. We propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.