bluuebunny/crossref_metadata_2025 huggingface dataset:
Toward Transplantation of Liver Organoids: From Biology and Ethics to Cost-effective Therapy
Authors: ten Dam Marjolein J.M. , Frederix Geert W.J. , ten Ham Renske M.T. , van der Laan Luc J.W. , Schneeberger Kerstin
Published: February 2023

Liver disease is a common cause of morbidity and mortality, and many patients would benefit from liver transplantation. However, because of a shortage of suitable donor livers, even of those patients who are placed on the donor liver waiting list, many do not survive the waiting time for transplantation. Therefore, alternative treatments for end-stage liver disease need to be explored. Recent advances in organoid technology might serve as a solution to overcome the donor liver shortage in the future. In this overview, we highlight the potential of organoid technology for cell therapy and tissue engineering approaches. Both organoid-based approaches could be used as treatment for end-stage liver disease patients. Additionally, organoid-based cell therapy can also be used to repair liver grafts ex vivo to increase the supply of transplantable liver tissue. The potential of both approaches to become clinically available is carefully assessed, including their clinical, ethical, and economic implications. We provide insight into what aspects should be considered further to allow alternatives to donor liver transplantation to be successfully clinically implemented.
Long-term Prolonged-release Tacrolimus-based Immunosuppression in De Novo Kidney Transplant Recipients: 5-Y Prospective Follow-up of Patients in the ADVANCE Study
Authors: Pernin Vincent , Glyda Maciej , Viklický Ondrej , Lõhmus Aleksander , Wennberg Lars , Witzke Oliver , von Zur-Mühlen Bengt , Anaokar Swapneel , Hurst Martin , Kazeem Gbenga , Undre Nasrullah , Kuypers Dirk R.J.
Published: February 2023

Background.

Although prolonged-release tacrolimus (PR-T) is widely approved for posttransplantation immunosuppression in kidney recipients, large-scale studies are required to assess long-term outcomes. We present follow-up data from the Advagraf-based Immunosuppression Regimen Examining New Onset Diabetes Mellitus in Kidney Transplant Recipients (ADVANCE) trial, in which kidney transplant patients (KTPs) received corticosteroid minimization with PR-T.

Methods.

ADVANCE was a 24-wk, randomized, open-label, phase-4 study. De novo KTPs received PR-T with basiliximab and mycophenolate mofetil and were randomized to receive an intraoperative corticosteroid bolus plus tapered corticosteroids until day 10 (arm 1) or an intraoperative corticosteroid bolus (arm 2). In this 5-y, noninterventional follow-up, patients received maintenance immunosuppression according to standard practice. The primary endpoint was graft survival (Kaplan-Meier). Secondary endpoints included patient survival, biopsy-confirmed acute rejection-free survival, and estimated glomerular filtration rate (4-variable modification of diet in renal disease).

Results.

Follow-up study included 1125 patients. Overall graft survival at 1 and 5 y posttransplantation was 93.8% and 88.1%, respectively, and was similar between treatment arms. At 1 and 5 y, patient survival was 97.8% and 94.4%, respectively. Five-year graft and patient survival rates in KTPs who remained on PR-T were 91.5% and 98.2%, respectively. Cox proportional hazards analysis demonstrated similar risk of graft loss and death between treatment arms. Five-year biopsy-confirmed acute rejection-free survival was 84.1%. Mean ± standard deviation values of estimated glomerular filtration rate were 52.7 ± 19.5 and 51.1 ± 22.4 mL/min/1.73 m2 at 1 and 5 y, respectively. Fifty adverse drug reactions were recorded, probably tacrolimus-related in 12 patients (1.5%).

Conclusions.

Graft survival and patient survival (overall and for KTPs who remained on PR-T) were numerically high and similar between treatment arms at 5 y posttransplantation.
Performance of a Prospective Anticoagulation Stratification Algorithm After Liver Transplantation
Authors: Sanchez-Garcia Jorge , Lopez-Verdugo Fidel , LeCorchick Spencer , Tran Alexandria , Gilroy Richard K. , Fujita Shiro , Zendejas Ivan , Gagnon Andrew , Dow Sean , Krong Jake , Rodriguez-Davalos Manuel I. , Stevens Scott M. , Woller Scott C. , Alonso Diane
Published: February 2023

Background.

Venous thromboembolism (VTE) occurs in 0.4% to 15.5% and bleeding occurs in 20% to 35% of patients after liver transplantation (LT). Balancing the risk of bleeding from therapeutic anticoagulation and risk of thrombosis in the postoperative period is challenging. Little evidence exists regarding the best treatment strategy for these patients. We hypothesized that a subset of LT patients who develop postoperative deep vein thromboses (DVTs) could be managed without therapeutic anticoagulation. We implemented a quality improvement (QI) initiative using a standardized Doppler ultrasound-based VTE risk stratification algorithm to guide parsimonious implementation of therapeutic anticoagulation with heparin drip.

Methods.

In a prospective management QI initiative for DVT management, we compared 87 LT historical patients (control group; January 2016–December 2017) to 182 LT patients (study group; January 2018–March 2021). We analyzed the rates of immediate therapeutic anticoagulation after DVT diagnosis within 14 d of LT, clinically significant bleeding, return to the operating room, readmission, pulmonary embolism, and death within 30 d of LT before and after the QI initiative.

Results.

Ten patients (11.5%) in the control group and 23 patients (12.6%; P = 0.9) in the study group developed DVTs after LT. Immediate therapeutic anticoagulation was used in 7 of 10 and 5 of 23 patients in the control and study groups, respectively (P = 0.024). The study group had lower odds of receiving immediate therapeutic anticoagulation after VTE (21.7% versus 70%; odds ratio = 0.12; 95% confidence interval, 0.019-0.587; P = 0.013) and a lower rate of postoperative bleeding (8.7% versus 40%; odds ratio = 0.14, 95% confidence interval, 0.02-0.91; P = 0.048). All other outcomes were similar.

Conclusions.

Implementing a risk-stratified VTE treatment algorithm for immediate post-LT patients appears to be safe and feasible. We observed a decrease in the use of therapeutic anticoagulation and a lower rate of postoperative bleeding without adverse impacts on early outcomes.
Genome-wide identification of lncRNAs associated with viral infection in Spodoptera frugiperda
Authors: Robin Stéphanie , Legeai Fabrice , Jouan Véronique , Ogliastro Mylène , Darboux Isabelle
Published: February 2023

The role of lncRNAs in immune defence has been demonstrated in many multicellular and unicellular organisms. However, investigation of the identification and characterization of long non-coding RNAs (lncRNAs) involved in the insect immune response is still limited. In this study, we used RNA sequencing (RNA-seq) to investigate the expression profiles of lncRNAs and mRNAs in the fall armyworm Spodoptera frugiperda in response to virus infection. To assess the tissue- and virus-specificity of lncRNAs, we analysed and compared their expression profiles in haemocytes and fat body of larvae infected with two entomopathogenic viruses with different lifestyles, i.e. the polydnavirus HdIV (Hyposoter didymator IchnoVirus) and the densovirus JcDV (Junonia coenia densovirus). We identified 1883 candidate lncRNAs, of which 529 showed differential expression following viral infection. Expression profiles differed considerably between samples, indicating that many differentially expressed (DE) lncRNAs showed virus- and tissue-specific expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and target prediction analyses indicated that DE-LncRNAs were mainly enriched in metabolic process, DNA replication and repair, immune response, metabolism of insect hormone and cell adhesion. In addition, we identified three DE-lncRNAs potentially acting as microRNA host genes, suggesting that they participate in gene regulation by producing miRNAs in response to virus infection. This study provides a catalogue of lncRNAs expressed in two important immune tissues and potential insight into their roles in the antiviral defence in S. frugiperda. The results may help future in-depth functional studies to better understand the biological function of lncRNAs in interaction between viruses and the fall armyworm.
Pathogenesis of wild-type- and vaccine-based recombinant peste des petits ruminants virus (PPRV) expressing EGFP in experimentally infected domestic goats
Authors: Schmitz Katharina S. , Eblé Phaedra L. , van Gennip René G. P. , Maris-Veldhuis Mieke A. , de Vries Rory D. , van Keulen Lucien J. M. , de Swart Rik L. , van Rijn Piet A.
Published: February 2023

Peste des petits ruminants virus (PPRV) is a highly contagious morbillivirus related to measles and canine distemper virus, mostly affecting small ruminants. The corresponding PPR disease has a high clinical impact in goats and is characterized by fever, oral and nasal erosions, diarrhoea and pneumonia. In addition, massive infection of lymphoid tissues causes lymphopaenia and immune suppression. This results in increased susceptibility to secondary bacterial infections, explaining the observed high mortality in some outbreaks. We studied the pathogenesis of PPR by experimental inoculation of Dutch domestic goats with a recombinant virulent PPRV strain modified to express EGFP and compared it to an EGFP-expressing vaccine strain of PPRV. After intratracheal inoculation with virulent PPRV, animals developed fever, viraemia and leucopaenia, and shed virus from the respiratory and gastro-intestinal tracts. Macroscopic evaluation of fluorescence at the peak of infection 7 days post-inoculation (dpi) showed prominent PPRV infection of the respiratory tract, lymphoid tissues, gastro-intestinal tract, mucosae and skin. Flow cytometry of PBMCs collected over time demonstrated a cell-associated viraemia mediated by infected lymphocytes. At 14 dpi, pathognomonic zebra stripes were detected in the mucosa of the large intestine. In contrast, vaccine strain-inoculated goats remained largely macroscopically fluorescence negative and did not present clinical signs. A low-level viraemia was detected by flow cytometry, but at necropsy no histological lesions were observed. Animals from both groups seroconverted as early as 7 dpi and sera efficiently neutralized virulent PPRVin vitro. Combined, this work presents a study of the pathogenesis of wild type- and vaccine-based PPRV in its natural host. This study shows the strength of recombinant EGFP-expressing viruses in fluorescence-guided pathogenesis studies.
One to host them all: genomics of the diverse bacterial endosymbionts of the spider Oedothorax gibbosus
Authors: Halter Tamara , Köstlbacher Stephan , Rattei Thomas , Hendrickx Frederik , Manzano-Marín Alejandro , Horn Matthias
Published: February 2023

Bacterial endosymbionts of the groups

Wolbachia

,

Cardinium

and

Rickettsiaceae

are well known for their diverse effects on their arthropod hosts, ranging from mutualistic relationships to reproductive phenotypes. Here, we analysed a unique system in which the dwarf spider Oedothorax gibbosus is co-infected with up to five different endosymbionts affiliated with

Wolbachia

, ‘Candidatus Tisiphia’ (formerly Torix group

Rickettsia

),

Cardinium

and

Rhabdochlamydia

. Using short-read genome sequencing data, we show that the endosymbionts are heterogeneously distributed among O. gibbosus populations and are frequently found co-infecting spider individuals. To study this intricate host–endosymbiont system on a genome-resolved level, we used long-read sequencing to reconstruct closed genomes of the

Wolbachia

, ‘Ca. Tisiphia’ and

Cardinium

endosymbionts. We provide insights into the ecology and evolution of the endosymbionts and shed light on the interactions with their spider host. We detected high quantities of transposable elements in all endosymbiont genomes and provide evidence that ancestors of the

Cardinium

, ‘Ca. Tisiphia’ and

Wolbachia

endosymbionts have co-infected the same hosts in the past. Our findings contribute to broadening our knowledge about endosymbionts infecting one of the largest animal phyla on Earth and show the usefulness of transposable elements as an evolutionary ‘contact-tracing’ tool.
The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo
Authors: Lau Alice C. C. , Mohamed Wessam Mohamed Ahmed , Nakao Ryo , Onuma Manabu , Qiu Yongjin , Nakajima Nobuyoshi , Shimozuru Michito , Mohd-Azlan Jayasilan , Moustafa Mohamed Abdallah Mohamed , Tsubota Toshio
Published: February 2023

Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between

Borrelia

-positive and

Borrelia

-negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as

Rickettsia heilongjiangensis

,

Ehrlichia

sp.,

Anaplasma

sp. and

Bartonella

spp. were characterized by PCR and sequencing. Moreover,

Coxiella

and

Francisella

-like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.
ChatGPT for Clinical Vignette Generation, Revision, and Evaluation
Authors: Benoit James R. A.
Published: February 2023

Objective

To determine the capabilities of ChatGPT for rapidly generating, rewriting, and evaluating (via diagnostic and triage accuracy) sets of clinical vignettes.

Design

We explored the capabilities of ChatGPT for generating and rewriting vignettes. First, we gave it natural language prompts to generate 10 new sets of 10 vignettes, each set for a different common childhood illness. Next, we had it generate 10 sets of 10 vignettes given a set of symptoms from which to draw. We then had it rewrite 15 existing pediatric vignettes at different levels of health literacy. Fourth, we asked it to generate 10 vignettes written as a parent, and rewrite these vignettes as a physician, then at a grade 8 reading level, before rewriting them from the original parent’s perspective. Finally, we evaluated ChatGPT for diagnosis and triage for 45 clinical vignettes previously used for evaluating symptom checkers.

Setting and participants

ChatGPT, a publicly available, free chatbot.

Main outcome measures

Our main outcomes for de novo vignette generation were whether ChatGPT followed vignette creation instructions consistently, correctly, and listed reasonable symptoms for the disease being described. For generating vignettes from pre-existing symptom sets, we examined whether the symptom sets were used without introducing extra symptoms. Our main outcome for rewriting existing standardized vignettes to match patient demographics, and rewriting vignettes between styles, was whether symptoms were dropped or added outside the original vignette. Finally, our main outcomes examining diagnostic and triage accuracy on 45 standardized patient vignettes were whether the correct diagnosis was listed first, and if the correct triage recommendation was made.

Results

ChatGPT was able to quickly produce varied contexts and symptom profiles when writing vignettes based on an illness name, but overused some core disease symptoms. It was able to use given symptom lists as the basis for vignettes consistently, adding one additional (though appropriate) symptom from outside the list for one disease. Pediatric vignettes rewritten at different levels of health literacy showed more complex symptoms being dropped when writing at low health literacy in 87.5% of cases. While writing at high health literacy, it added a diagnosis to 80% of vignettes (91.7% correctly diagnosed). Symptoms were retained in 90% of cases when rewriting vignettes between viewpoints. When presented with 45 vignettes, ChatGPT identified illnesses with 75.6% (95% CI, 62.6% to 88.5%) first-pass diagnostic accuracy and 57.8% (95% CI, 42.9% to 72.7%) triage accuracy. Its use does require monitoring and has caveats, which we discuss.

Conclusions

ChatGPT was capable, with caveats and appropriate review, of generating, rewriting, and evaluating clinical vignettes.
Individual costs and societal benefits of interventions during the COVID-19 pandemic
Authors: Traulsen Arne , Levin Simon A. , Saad-Roy Chadi M.
Published: February 2023

Individual and societal reactions to an ongoing pandemic can lead to social dilemmas: In some cases, each individual is tempted to not follow an intervention, but for the whole society it would be best if they did. Now that in most countries the extent of regulations to reduce SARS-CoV-2 transmission is very small, interventions are driven by individual decision-making. Assuming that individuals act in their best own interest, we propose a framework in which this situation can be quantified, depending on the protection the intervention provides to a user and to others, the risk of getting infected, and the costs of the intervention. We discuss when a tension between individual and societal benefits arises and which parameter comparisons are important to distinguish between different regimes of intervention use.
Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality
Authors: Shi Tiffany , Burg Ashley R. , Caldwell J. Timothy , Roskin Krishna , Castro-Rojas Cyd M. , Chukwuma P. Chukwunalu , Gray George I. , Foote Sara G. , Alonso Jesus , Cuda Carla M. , Allman David A. , Rush James S. , Regnier Catherine H. , Wieczorek Grazyna , Alloway Rita R. , Shields Adele R. , Baker Brian M. , Woodle E. Steve , Hildeman David A.
Published: February 2023

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/β sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8+T cell clonal expansion (CD8EXP), independent of HLA mismatch or IS type. Subcloning of TCRα/β cDNAs from CD8EXPinto Jurkat76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8EXPrevealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8EXP, while CD8EXPwere maintained during treatment-refractory rejection. Finally, most rBx-derived CD8EXPwere also observed in matching urine samples. Overall, our data define the clonal CD8+T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.
Insights intoTreponema pallidumgenomics from modern and ancient genomes using a novel mapping strategy
Authors: Pla-Díaz Marta , Akgül Gülfirde , Molak Martyna , du Plessis Louis , Panagiotopoulou Hanna , Doan Karolina , Bogdanowicz Wiesław , Dąbrowski Paweł , Oziębłowski Maciej , Kwiatkowska Barbara , Szczurowski Jacek , Grzelak Joanna , Arora Natasha , Majander Kerttu , González-Candelas Fernando , Schuenemann Verena J.
Published: February 2023

Background

Treponemal diseases pose significant global health risks, presenting severe challenges to public health due to their serious health impacts if left untreated. Despite numerous genomic studies onTreponema pallidumand the known possible biases introduced by the choice of the reference genome used for mapping, few investigations have addressed how these biases affect phylogenetic and evolutionary analysis of these bacteria. In this study, we assessed the impact of selecting an appropriate genomic reference on phylogenetic and evolutionary analyses ofT. pallidum.

Results

We designed a multiple-reference-based (MRB) mapping strategy using four different reference genomes and compared it to traditional single-reference mapping. To conduct this comparison, we created a genomic dataset comprising 77 modern and ancient genomes from the three subspecies ofT. pallidum, including a newly sequenced 17th-century genome (35X coverage) of a syphilis-causing strain (designated as W86). Our findings show that recombination detection was consistent across different references, but the choice of reference significantly affected ancient genome reconstruction and phylogenetic inferences. The high-coverage W86 genome obtained here also provided a new calibration point for Bayesian molecular clock dating, improving the reconstruction of the evolutionary history of treponemal diseases. Additionally, we identified novel recombination events, positive selection targets, and refined dating estimates for key events in the species’ history.

Conclusions

This study highlights the importance of considering methodological implications and reference genome bias in High-Throughput Sequencing-based whole-genome analysis ofT. pallidum, especially of ancient or low-coverage samples, contributing to a deeper understanding of this pathogen and its subspecies.
The parabrachial to central amygdala circuit is a key mediator of injury-induced pain sensitization
Authors: Torres-Rodriguez Jeitzel M. , Wilson Torri D. , Singh Sudhuman , Chaudhry Sarah , Adke Anisha P. , Becker Jordan J. , Lin Jenny L. , Gonzalez Santiago Martinez , Soler-Cedeño Omar , Carrasquillo Yarimar
Published: February 2023

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PbN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. At the behavioral level, the PbN→CeA pathway has been proposed to serve as a general alarm system to potential threats that modulates pain-related escape behaviors, threat memory, aversion, and affective-motivational (but not somatosensory) responses to painful stimuli. Increased sensitivity to previously innocuous somatosensory stimulation is a hallmark of chronic pain. Whether the PbN→CeA circuit contributes to heightened peripheral sensitivity following an injury, however, remains unknown. Here, we demonstrate that activation of CeA-projecting PbN neurons contributes to injury-induced behavioral hypersensitivity but not baseline nociception in male and female mice. Using optogenetic assisted circuit mapping, we confirmed a functional excitatory projection from PbN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increases the expression of the neuronal activity marker c-Fos in CeA-projecting PbN neurons and chemogenetic inactivation of these cells reduces behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we show that chemogenetic activation of CeA-projecting PbN neurons is sufficient to induce bilateral hypersensitivity without injury. Together, our results demonstrate that the PbN→CeA pathway is a key modulator of pain-related behaviors that can amplify responses to somatosensory stimulation in pathological states without affecting nociception under normal physiological conditions.

Significance Statement

Early studies identified the spino-ponto-amygdaloid pathway as a major ascending circuit conveying nociceptive inputs from the spinal cord to the brain. The functional significance of this circuit to injury-induced hypersensitivity, however, remains unknown. Here, we addressed this gap in knowledge using viral-mediated anatomical tracers,ex-vivoelectrophysiology and chemogenetic intersectional approaches in rodent models of persistent pain. We found that activation of this pathway contributes to injury-induced hypersensitivity, directly demonstrating a critical function of the PbN→CeA circuit in pain modulation.
Inverse-folding design of yeast telomerase RNA increases activityin vitro
Authors: Lebo Kevin J. , Zappulla David C.
Published: February 2023

Saccharomyces cerevisiaetelomerase RNA, TLC1, is an 1157 nt non-coding RNA that functions as both a template for DNA synthesis and a flexible scaffold for telomerase RNP holoenzyme protein subunits. The tractable budding yeast system has provided landmark discoveries about telomere biologyin vivo, but yeast telomerase research has been hampered by the fact that the large TLC1 RNA subunit does not support robust telomerase activityin vitro. In contrast, 155–500 nt miniaturized TLC1 alleles comprising the catalytic core domain and lacking the RNA’s long arms do reconstitute robust activity. We hypothesized that full-length TLC1 is prone to misfoldingin vitro. To create a full-length yeast telomerase RNA predicted to fold into its biological relevant structure, we took an inverse RNA folding approach, changing 59 nucleotides predicted to increase the energetic favorability of folding into the modeled native structure based on thep-numfeature ofMfoldsoftware. The sequence changes lowered the predicted ∆G in this “determined-arm” allele, DA-TLC1, by 61 kcal/mol (–19%) compared to wild type. We tested DA-TLC1 for reconstituted activity and found it to be ∼5-fold more robust than wild-type TLC1, suggesting that the inverse-folding design indeed improved foldingin vitrointo a catalytically active conformation. We also tested if DA-TLC1 functionsin vivoand found that it complements atlc1∆ strain, allowing cells to avoid senescence and maintain telomeres of nearly wild-type length. However, all inverse-designed RNAs that we tested had reduced abundancein vivo. In particular, inverse-designing nearly all of the Ku arm caused a profound reduction in telomerase RNA abundance in the cell and very short telomeres. Overall, these results show that inverse design ofS. cerevisiaetelomerase RNA increases activityin vitro, while reducing abundancein vivo. This study provides a biochemically and biologically tested approach to inverse-design RNAs usingMfoldthat could be useful for controlling RNA structure in basic research and biomedicine.
Exceeding 80% efficiency of single-bead encapsulation in microdroplets through hydrogel coating-assisted close-packed ordering
Authors: Chen Long , Zhao Yi , Li Jie , Xiong Chenwei , Xu Yi , Tang Chengren , Zhang Rong , Zhang Jingwei , Mi Xianqiang , Liu Yifan
Published: February 2023

High-efficiency encapsulation of single microbeads in microdroplets is essential for droplet-based high-throughput analysis such as single-cell genomics and digital immunoassays. However, the demand has been hindered by the Poisson statistics of beads arbitrarily distributed in the droplet partitions. Although techniques such as inertial ordering have been proven useful to improve bead loading efficiency, a general method that requires no advanced microfluidics and owns compatibility with diverse bead types is still highly desired. In this paper, we present hydrogel coating-assisted close-packed ordering, a simple strategy that improves the bead loading efficiency to over 80%. In the strategy, the raw beads are coated with a thin layer of hydrogel to become slightly compressible and lubricious so that they can be close-packed in a microfluidic device and loaded into droplets in a synchronized manner. We first show that the thin hydrogel coating can be realized conveniently through either jetting microfluidics or vortex emulsification. When loading single 30-μm polystyrene beads, we experimentally determined an overall efficiency of 81% with the proposed hydrogel coating strategy. Of note, the strategy is not sensitive to the selection of raw beads and can tolerate their polydispersity. Using the strategy, we achieve a cell capture rate of 68.8% when co-encapsulating HEK293T cells and polydispersed barcoded beads for single-cell transcriptomics. Further sequencing results verify that the reversible hydrogel coating does not affect the RNA capture behavior of the encapsulated barcoded beads. Given its convenience and broad compatibility, we anticipate that our strategy can be applied to various droplet-based high-throughput assays to drastically improve their efficiency.
Extending species-area relationships into the realm of ecoacoustics: The soundscape-area relationship
Authors: Luypaert Thomas , Bueno Anderson S. , Haugaasen Torbjørn , Peres Carlos A.
Published: February 2023

The rise in species richness with area is one of the best-studied patterns in ecology. Yet, little is known about the spatial scaling of alternate dimensions of diversity. Here, we provide empirical evidence for a relationship between the richness of acoustic traits emanating from the landscape, or soundscape richness, and the island area, which we term the Soundscape Area Relationship (SSAR). We show a positive relationship between the gamma soundscape richness and island area, with slope and R2-values of comparable magnitude to those reported in the literature. This relationship breaks down at the smallest spatial scales, indicating a small-island effect. Moreover, we demonstrate a positive spatial scaling of the plot-scale alpha soundscape richness, but not the beta soundscape turnover, suggesting disproportionate effects are an important underlying mechanism. We conclude that the scaling of biodiversity can be extended to the realm of ecoacoustics, implying soundscape metrics are sensitive to fundamental ecological patterns and useful to disentangle the complex mechanisms that drive them.
A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins
Authors: Rex Emily A. , Seo Dahee , Chappidi Sruthi , Pinkham Chelsea , Oliveira Sabrynna Brito , Embry Aaron , Heisler David , Liu Yang , Luger Karolin , Alto Neal M. , da Fonseca Flávio Guimarães , Orchard Robert , Hancks Dustin , Gammon Don B.
Published: February 2023

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals theFACT-ETS-1AntiviralResponse (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.
Real-time spectral library matching for sample multiplexed quantitative proteomics
Authors: McGann Chris D. , Barshop Will , Canterbury Jesse , Lin Chuwei , Gabriel Wassim , Wilhelm Mathias , McAlister Graeme , Schweppe Devin K.
Published: February 2023

Sample multiplexed quantitative proteomics has proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor co-isolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Towards this end, we sought to develop and implement a new real-time library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time as traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy in sample multiplexed analyses.
Breast adipose tissue-derived extracellular vesicles from women with obesity stimulate mitochondrial-induced dysregulated tumor cell metabolism
Authors: Liu Shuchen , Benito-Martin Alberto , Pelissier Vatter Fanny A. , Hanif Sarah Z. , Liu Catherine , Bhardwaj Priya , Sethupathy Praveen , Farghli Alaa R. , Piloco Phoebe , Paik Paul , Mushannen Malik , Otterburn David M. , Cohen Leslie , Bareja Rohan , Krumsiek Jan , Cohen-Gould Leona , Calto Samuel , Spector Jason A. , Elemento Olivier , Lyden David , Brown Kristy A.
Published: February 2023

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Dysregulated cell metabolism is now an accepted hallmark of cancer. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we found that long-term education of breast cancer cells (MCF7, T47D) with EVs from breast adipose tissue of women who are overweight or obese (O-EVs) leads to sustained increased proliferative potential. RNA-Seq of O-EV-educated cells demonstrates increased expression of genes, such as ATP synthase and NADH: ubiquinone oxidoreductase, involved in oxidative phosphorylation. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. Mitochondrial complex I inhibitor, metformin, reverses O-EV-induced cell proliferation. Several miRNAs, miR-155-5p, miR-10a-3p, and miR-30a-3p, which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing the metabolic reprogramming of ER+ breast cancer cells.
Does Music Training Improve Inhibition Control in Children? A Systematic Review and Meta-Analysis
Authors: Jamey Kevin , Foster Nicholas E. V. , Hyde Krista L. , Dalla Bella Simone
Published: February 2023

Inhibition control is an essential executive function during children’s development, underpinning self-regulation and the acquisition of social and language abilities. This executive function is intensely engaged in music training while learning an instrument, a complex multisensory task requiring monitoring motor performance and auditory stream prioritization. This novel meta-analysis examined music-based training on inhibition control in children. Records from 1980 to 2023 yielded 22 longitudinal studies with controls (N = 1734), including 8 RCTs and 14 others. A random-effects meta-analysis showed that music training improved inhibition control (medium ES) in the RCTs and the superset of twenty-two longitudinal studies (small-to-medium ES). Music training plays a privileged role compared to other activities (sports, visual arts, drama) in improving children’s executive functioning, with a particular effect on inhibition control. We recommend music training for complementing education and as a clinical tool focusing on inhibition control remediation (e.g., in autism and ADHD).
Signal Integration and Adaptive Sensory Diversity Tuning inEscherichia coliChemotaxis
Authors: Moore Jeremy Philippe , Kamino Keita , Kottou Rafaela , Shimizu Thomas , Emonet Thierry
Published: February 2023

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal ambient concentration increases. However, amongst competitive ligands the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefitsE. colipopulations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.
LINKIN-associated proteins necessary for tissue integrity during collective cell migration
Authors: Tan Chieh-Hsiang , Cheng Kai-Wen , Park Heenam , Chou Tsui-Fen , Sternberg Paul W.
Published: February 2023

Cell adhesion plays essential roles in almost every aspect of metazoan biology. LINKIN (Human: ITFG1,Caenorhabditis elegans:lnkn-1) is a conserved transmembrane protein that has been identified to be necessary for tissue integrity during migration. InC. elegans, loss oflnkn-1results in the detachment of the lead migratory cell from the rest of the developing male gonad. Previously, three interactors of ITFG1/lnkn-1– RUVBL1/ruvb-1, RUVBL2/ruvb-2, and alpha-tubulin – were identified by immunoprecipitation-mass spectrometry (IP-MS) analysis using human HEK293T cells and then validated in the nematode male gonad. The ITFG1-RUVBL1 interaction has since been independently validated in a breast cancer cell line model that also implicates the involvement of the pair in metastasis. Here, we showed that epitope-tagged ITFG1 localized to the cell surface of MDA-MB-231 breast cancer cells. Using IP-MS analysis, we identified a new list of potential interactors of ITFG1.Loss-of-functionanalysis of theirC. elegansorthologs found that three of the interactors – ATP9A/tat-5, NME1/ndk-1, and ANAPC2/apc-2– displayed migratory detachment phenotypes similar to that oflnkn-1. Taken together with the other genes whose reduction-of-function phenotype is similar to that oflnkn-1(notably cohesion and condensin), suggests the involvement of membrane remodeling and chromosome biology in LINKIN-dependent cell adhesion and supports the hypothesis for a structural role of chromosomes in post-mitotic cells.
Spotty distributions: Spotted Gar (Lepisosteus oculatus) and Spotted Sucker (Minytrema melanops) range expansion in eastern Lake Erie
Authors: MacGuigan Daniel J. , Porto-Hannes Isabel , Foote Brian , Backenstose Nathan J. C. , Osborne Christopher , Louisor Kimberly , Waterman Hannah , Chang Sarah L. , Cochran Jacob L. , Krabbenhoft Trevor J.
Published: February 2023

Natural range expansions in warm-water freshwater fishes are currently not well understood, but shifts in native species distributions can be influenced by many factors, including habitat restoration or degradation and climate change. Here, we provide empirical evidence of range expansions observed in two native freshwater fish species in Lake Erie: the Spotted Gar (Lepisosteus oculatus)and Spotted Sucker (Minytrema melanops). We confirmed our field identifications ofL. oculatusandM. melanopsusing mtDNA barcoding. Maximum likelihood phylogenetic analyses reveal that our samples confidently resolve in theL. oculatusandM. melanopsclades respectively, with additional identification support from BLAST searches. Notably, we found no correlation between the increased detection rate of both species and an increase in sampling effort when compared to previous records. Historically, eastern Lake Erie experienced habitat degradation through channelization, siltation, dredging, and toxification of sediments. We hypothesize that recent habitat remediation efforts have provided suitable habitat for both species to recolonize shallow waters with densely vegetated habitat (>90% substrate coverage). Both species are likely to continue their northern expansion as habitats are restored and climatic changes favor warm-water fishes.
The origins and diversification of Holarctic brown bear populations inferred from genomes of past and present populations
Authors: Segawa Takahiro , Rey-Iglesia Alba , Lorenzen Eline D , Westbury Michael V
Published: February 2023

The brown bear (Ursus arctos) is one of the survivors of the Late Quaternary megafauna extinctions. However, despite being widely distributed across the Holarctic, brown bears have experienced extensive range reductions, and even extirpations in some geographic regions. Previous research efforts utilising genetic data have provided valuable insights into their evolutionary history. However, most studies have been limited to contemporary individuals or mitochondrial DNA, limiting insights into population processes that preceded the present. Here we present genomic data from two Late Pleistocene brown bears from Honshu, Japan, and eastern Siberia, and combine them with published contemporary and ancient genomes from across the Holarctic range of brown bears to investigate the evolutionary relationships among brown bear populations through time and space. By including genomic data from Late Pleistocene and Holocene individuals sampled outside the current distribution range we uncover diversity not present in the contemporary populations. Notably, although contemporary individuals display geographically structured populations most likely driven by isolation-by-distance, this pattern varies among the ancient samples across different regions. The inclusion of ancient brown bears in our analysis provides novel insights into the evolutionary history of brown bears and contributes to understanding the populations and diversity lost during the Late Quaternary.
Root associated bacterial communities and root metabolite composition are linked to nitrogen use efficiency in sorghum
Authors: Chai Yen Ning , Qi Yunhui , Goren Emily , Sheflin Amy M. , Tringe Susannah , Prenni Jessica E. , Liu Peng , Schachtman Daniel
Published: February 2023

Development of cereal crops with high nitrogen-use efficiency (NUE) is a priority for worldwide agriculture. In addition to conventional plant breeding and genetic engineering, the use of the plant microbiome offers another approach to improve crop NUE. To gain insight into the bacterial communities associated with sorghum lines that differ in NUE, a field experiment was designed comparing 24 diverse sorghum lines under sufficient and deficient nitrogen (N). Amplicon sequencing and untargeted gas chromatography-mass spectrometry (GC-MS) were used to characterize the bacterial communities and the root metabolome associated with sorghum genotypes varying in sensitivity to low N. We demonstrated that N stress and sorghum type (energy, sweet, and grain sorghum) significantly influenced the root-associated bacterial communities and root metabolite composition of sorghum. Sorghum NUE was positively correlated with the bacterial richness and diversity in the rhizosphere. The greater alpha diversity in high NUE lines was associated with the decreased abundance of a dominant bacterial taxa,Pseudomonas. Multiple strong correlations were detected between root metabolites and rhizosphere bacterial communities in response to N stress and indicate that the shift in the sorghum microbiome due to low-N is associated with the root metabolites of the host plant. Taken together, our study provides new insight into the links between host genetic regulation of root metabolites and root-associated microbiome of sorghum genotypes differing in NUE and tolerance to low-N stress.
Pre- and postsynaptic nanostructures increase in size and complexity after LTP induction
Authors: Clavet-Fournier Valérie , Lee ChungKu , Wegner Waja , Brose Nils , Rhee JeongSeop , Willig Katrin I.
Published: February 2023

Synapses, specialized contact sites between neurons, are the fundamental elements of neuronal information transfer. Synaptic plasticity is related to changes in synaptic morphology and the number of neurotransmitter receptors, and thought to underlie learning and memory. However, it is not clear how these structural and functional changes are connected. We utilized time-lapse super-resolution STED microscopy to visualize structural changes of the synaptic nano-organization of the postsynaptic scaffolding protein PSD95, the presynaptic scaffolding protein Bassoon, and the GluA2 subunit of AMPA receptors by chemically induced long-term potentiation (cLTP) at the level of single synapses. We found that the nano-organization of all three proteins undergoes an increase in complexity and size after cLTP induction. The increase was largely synchronous, peaking at ∼60 min after stimulation. Therefore, both the size and complexity of single pre- and post-synaptic nanostructures serve as substrates for adjusting and determining synaptic strength.

Highlights

- Time-lapse super-resolution images the structural changes of the PSD95 nano-organization after Cltp

- cLTP-induced growth of the PSD95 nano-organization is less than spine head growth and peaks at 60 min, i.e. much slower than the increase in spine volume.

- Most PSD95 nanostructures increase in complexity upon cLTP.

- Synchronous growth - Nanostructures of pre- and postsynaptic scaffolding proteins and AMPA receptors increase simultaneously and equally strong upon cLTP.

- GluA2-containing synaptic AMPA receptors form nanoclusters that increase in size and slightly in number upon cLTP and form subdomains on PSD95.

- Bassoon forms complex structures similar to the PSD95 nano-organization.