bluuebunny/crossref_metadata_2025 huggingface dataset:
Gustatory-neuron-supplied R-spondin-2 is required for taste bud replenishment
Authors: Xu Jiang , de Araujo Alan Moreira , Xi Ranhui , Lin Xiaoli , Lu Chanyi , Zhou Minliang , Hankenson Kurt , Margolskee Robert F. , Matsumoto Ichiro , de Lartigue Guillaume , Choi Myunghwan , Jiang Peihua
Published: February 2024

Taste buds undergo continuous cell turnover throughout life, and taste cell replenishment relies strictly on innervation, a phenomenon first described almost 150 years ago. Recently, we provided evidence that R-spondin 2 (Rspo2) may be the long-sought gustatory neuron-supplied factor that regulates taste stem cell activity, via its interaction with taste stem/progenitor cell-expressed receptor Rnf43/Znrf3. Yet, whether gustatory-neuron-supplied Rspo2 is strictly required for taste tissue maintenance has not been resolved. Here, we set out to determine the necessity of gustatory-neuron-supplied Rspo2 in taste tissue homeostasis using genetic approaches. We used a mouse line that harbors the neomycin-resistance gene (NeoR) in one of the intron regions of theRspo2gene, which results in reduced expression of Rspo2. The number of taste buds is significantly reduced in these mice, compared to wild-type mice, in both anterior and posterior tongue. This phenotypic change was completely reversed by removingNeoRfrom theRspo2gene, thus making it normal. We also combined adeno-associated virus (AAV)-based delivery of Cre recombinase with a mouse line amenable to Cre-based ablation of theRspo2exons encoding the receptor-binding domains. Such deletion of Rspo2 in the nodose-petrosal-jugular ganglion complex led to nearly complete loss of taste buds in the circumvallate papilla. Thus, we demonstrate that Rspo2 is the long-sought gustatory-neuron-supplied factor that acts on taste stem cells to maintain taste tissue homeostasis.

Significance

We have known for 150 years that innervation is required to induce and maintain cell replacement in taste buds. Until recently, the identity of the inducing factor produced by neurons was unknown. We have shown that R-spondin alone is sufficient to substitute for neuronal input to induce taste bud regeneration. Using a genetic loss-of-function approach, we now demonstrate that gustatory-neuron-expressed Rspo2 is required to maintain taste tissue homeostasis. Altogether, our work reveals that Rspo2 is the long-sought neuron-supplied factor that regulates the activity of taste stem/progenitor cells.
CNVeil enables accurate and robust tumor subclone identification and copy number estimation from single-cell DNA sequencing data
Authors: Yuan Weiman , Luo Can , Hu Yunfei , Zhang Liting , Wen Zihang , Liu Yichen Henrry , Mallory Xian , Zhou Xin Maizie
Published: February 2024

Single-cell DNA sequencing (scDNA-seq) has significantly advanced cancer research by enabling precise detection of chromosomal aberrations, such as copy number variations (CNVs), at a single-cell level. These variations are crucial for understanding tumor progression and heterogeneity among tumor subclones. However, accurate CNV inference in scDNA-seq has been constrained by several factors, including low coverage, sequencing errors, and data variability. To address these challenges, we introduce CNVeil, a robust quantitative algorithm designed to accurately reveal CNV profiles while overcoming the inherent noise and bias in scDNA-seq data. CNVeil incorporates a unique bias correction method using normal cell profiles identified by a PCA-based Gini coefficient, effectively mitigating sequencing bias. Subsequently, a multi-level hierarchical clustering, based on selected highly variable bins, is employed to initially identify coarse subclones for robust ploidy estimation and further identify fine subclones for segmentation. To infer the CNV segmentation landscape, a novel change rate-based across-cell breakpoint identification approach is specifically designed to diminish the effects of low coverage and data variability on a per-cell basis. Finally, a consensus segmentation is utilized to further standardize read depth for the inference of the final CNV profile. In comprehensive benchmarking experiments, where we compared CNVeil with seven state-of-the-art CNV detection tools, CNVeil exhibited exceptional performance across a diverse set of simulated and real scDNA-seq data in cancer genomics. CNVeil excelled in subclone identification, segmentation, and CNV profiling. In light of these results, we anticipate that CNVeil will significantly contribute to single-cell CNV analysis, offering enhanced insights into chromosomal aberrations and genomic complexity.
Ephrin-B2 promotes nociceptive plasticity and hyperalgesic priming through EphB2-MNK-eIF4E signaling in both mice and humans
Authors: David Eric T. , Yousuf Muhammad Saad , Mei Hao-Ruei , Jain Ashita , Krishnagiri Sharada , Srikanth Kolluru D. , Dussor Gregory , Dalva Matthew B. , Price Theodore J.
Published: February 2024

Ephrin-B-EphB signaling promotes pain through signaling between dorsal root ganglion (DRG) neurons and spinal cord neurons in the dorsal horn, and through signaling between peripheral cells and EphB receptors expressed by DRG neurons. Previous findings link ephrin-B expression in painful peripheral tissues in patients to chronic pain, suggesting the clinical significance of this signaling, but the direct effects of ephrins on DRG neurons have not been widely studied. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Our work demonstrates that ephrin-B2-EphB2 signaling drives activation of MNK-eIF4E in DRG neurons to cause an enhanced response to inflammatory mediator signaling in both mice and humans and hyperalgesic priming in two models in mice. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2injection into the same hindpaw. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1knockout mice) and by the MNK inhibitor eFT508. Similar effects on hyperalgesic priming were seen in a dural injection model. We generated a sensory neuron specific knockout of EphB2 using Pirt-Cre mice and found that these mice lacked responses to ephrin-B2 injection. We used Ca2+-imaging to determine direct effects of ephrin-B2 on DRG neurons and found that ephrin-B2 treatment enhanced Ca2+transients in response to PGE2which were absent in DRG neurons from MNK1−/−and EphB2-PirtCremice. In experiments on human DRG neurons we found that ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+responses to PGE2treatment, both of which were blocked by eFT508 treatment. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling. The findings offer insight into how ephrin-B signaling promotes pain, and suggests treatment avenues for prevention or reversal of chronic pain associated with EphB activation in sensory neurons.

Graphical Abstract
FSTL1 is an antagonist of ERK1/2 phosphorylation during ciliogenesis and preadipocyte differentiation
Authors: Santos Leonardo , Guggeri Lucia , Escande Carlos , Badano José Luis , Prieto-Echagüe Victoria
Published: February 2024

FSTL1 is a secreted glycoprotein that is involved in several processes in health and disease, including development, cardiovascular disease, cancer, inflammation, and obesity. The signaling pathways used by FSTL1 to act on target tissues seem to activate different intracellular mediators specific to each tissue and several of the mechanisms of action remain to be determined at the molecular level, including intracellular mediators and receptors. We have previously unveiled a novel role for FSTL1 in ciliogenesis and provided evidence for an Fstl1/cilia axis in preadipocyte differentiation. This pathway is relevant to the pathogenesis of obesity and of a group of conditions called ciliopathies since they are caused by the dysfunction of the primary cilia. This work aimed to identify intracellular mediators of FSTL1 action on ciliogenesis and adipogenesis. We analyzed ERK phosphorylation levels as well as cilia length in the absence of FSTL1 and in the presence of the pERK inhibitor U0126. We also analyzed the differentiation and cilia dynamics of U0126-treated preadipocytes and tested the ERK-mediated signaling by BMP4 in the presence of added extracellular Fstl1. Here, we propose that MAP kinase ERK is a mediator of ciliogenesis downstream of FSTL1 and provide additional data that suggest that FSTL1 antagonizes BMP non-canonical signaling to modulate ciliogenesis and adipogenesis. In sum, our data reinforce the interest on the axis FSTL1/cilia in the modulation of adipogenesis and provide evidence to add ERK to this working model.
Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios
Authors: Weakly Heidi M.J. , Wilson Kent J. , Goetz Gunnar J. , Pruitt Emily L. , Li Amy , Xu Libin , Keller Sarah L.
Published: February 2024

Researchers choose different methods of making giant unilamellar vesicles in order to satisfy different constraints of their experimental designs. A challenge of using a variety of methods is that each may produce vesicles of different lipid compositions, even if all vesicles are made from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicles made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either 5-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts (≤ 5 mol%) in lipid ratios of vesicles relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of some of the lipids largely support results from the 5-component mixture. Exact values of lipid ratios variations likely depend on the details of each method, so a broader conclusion is that experiments that increment lipid ratios in small steps will be highly sensitive to the method of lipid formation and to sample-to-sample variations, which are low (roughly ±2 mol% in the 5-component mixture and either scale proportionally with increasing mole fraction or remain low). Experiments that increment lipid ratios in larger steps or that seek to explain general trends or new phenomena will be less sensitive to the method used.

SIGNIFICANCE STATEMENT

Small changes to the amounts and types of lipids in membranes can drastically affect the membrane’s behavior. Unfortunately, it is unknown whether (or to what extent) different methods of making vesicles alter the ratios of lipids in membranes, even when identical stock solutions are used. This presents challenges for researchers when comparing data with colleagues who use different methods. Here, we measure ratios of lipid types in vesicle membranes produced by five methods. We assess each method’s reproducibility and compare resulting vesicle compositions across methods. In doing so, we provide a quantitative basis that the scientific community can use to estimate whether differences between their results can be simply attributed to differences between methods or to sample-to-sample variations.
The influence of environment on mosquito feeding patterns: a meta-analysis of ‘universal’ DNA diet studies in a global context
Authors: O′Rorke Richard , Lee Meshach , Clark Nicholas J. , Webster Tamsyn Uren , Wells Konstans
Published: February 2024

Mosquitoes have innate preferences for their blood-meal hosts, but these can be modified by the environment, with implications for disease spread under climate and land use change. To predict the spread of vector-borne pathogens more accurately we need to better understand blood-meal plasticity under changing environmental conditions. We compiled blood-meal studies for six prominent disease vectoring mosquitoes from around the globe (Aedes aegypti, Ae. albopictus,Anopheles funestus, An. gambiae,Cx. pipiens, and Cx. quinquefasciatus). We targeted studies that used universal molecular methods (typically PCR/metabarcoding) to identify hosts from a wide range of candidates (as opposed to studies using methods that presuppose host identity - such as precipitin, ELISA). We found that blood-meals from the >15,600 analysed were mostly from the expected host-groups for each mosquito species, but we frequently encountered atypical hosts (e.g. mammalophilic species feeding on birds/reptiles). The universal methods used by the studies in our metanaalysis identified high host richness, and we found ≥174 hosts forCulexand ≤65 species forAedesmosquitoes at a considerably increased discovery rate of novel hosts per sampling effort. We used a hierarchical Dirichlet regression model to analyse global variation in feeding patterns in relation to environmental datasets (land use, precipitation, mean annual temperature, latitude, human and livestock density). Land use, mean annual temperature and poultry density had noticeable effects on blood-meal selection ofAe. aegypti,Cx. pipiensandCx. quinquefasciatus. Although host density was a factor in blood-meal selection - host choice is not a simple function of host availability, as has previously been observed, but contingent on other drivers. While our compiled dataset afforded us these insights, improving resolution and consistency of data gathering and reporting would improve the precision of how blood-meal studies can inform us of present and potential risks of pathogen transmission events.
Cephalopod Sex Determination and its Ancient Evolutionary Origin
Authors: Coffing Gabrielle C. , Tittes Silas , Small Scott T. , Songco-Casey Jeremea O. , Piscopo Denise M. , Pungor Judit R. , Miller Adam C. , Niell Cristopher M. , Kern Andrew D.
Published: February 2024

Octopuses, squids, and cuttlefishes – the coleoid cephalopods – are a remarkable branch in the tree of life whose members exhibit a repertoire of sophisticated behaviors (Hanlon and Messenger, 2018). As a clade, coleoids harbor an incredible variety of novel traits including the most complex nervous system among invertebrates, derived camera-type eyes, and rapid adaptive camouflage abilities (Young, 1971; Hanlon, 2007). The burst of evolutionary novelty that distinguishes cephalopods is even more striking in a phylogenetic context; cephalopods are a deeply diverged lineage that last share a common ancestor with other extant molluscs in the Cambrian period, roughly 550 million years ago (Ponder and Lindberg, 2008; Huang et al., 2022). With recent advances in genome sequencing technologies, we have the capability to explore the genomic foundations of cephalopod novelties. Here, using PacBio long-read sequencing of genomic DNA and IsoSeq full-length mRNA sequencing, we provide a novel chromosome-scale reference genome and annotation for a female California two-spot octopus (O. bimaculoides). Our assembly reveals that the female octopus has just one sex chromosome, consistent with a ZO karyotype, while the male has two (ZZ), providing the first evidence of genetic sex determination in cephalopods. We use our assembly and annotation in combination with existing genomic information from other cephalopods to create the first whole genome alignments from this group and demonstrate that the sex chromosome is of an ancient origin, before the radiation of extant cephalopods approximately 480 million years ago (Huang et al., 2022), and has been conserved to the present day in all cephalopod genomes available.
Spherical Echo-Planar Time-resolved Imaging (sEPTI) for rapid 3D quantitative T2* and Susceptibility imaging
Authors: Wang Nan , Liao Congyu , Cao Xiaozhi , Nishimura Mark , Brackenier Yannick WE , Yurt Mahmut , Gao Mengze , Abraham Daniel , Alkan Cagan , Iyer Siddharth Srinivasan , Zhou Zihan , Kerr Adam , Haldar Justin P. , Setsompop Kawin
Published: February 2024

Purpose

To develop a 3D spherical EPTI (sEPTI) acquisition and a comprehensive reconstruction pipeline for rapid high-quality whole-brain submillimeter T2* and QSM quantification.

Methods

For the sEPTI acquisition, spherical k-space coverage is utilized with variable echo-spacing and maximum kxramp-sampling to improve efficiency and incoherency when compared to existing EPTI approaches. For reconstruction, an iterative rank-shrinking B0estimation and odd-even high-order phase correction algorithms were incorporated into the reconstruction to better mitigate artifacts from field imperfections. A physics-informed unrolled network was utilized to boost the SNR, where 1-mm and 0.75-mm isotropic whole-brain imaging were performed in 45 and 90 seconds, respectively. These protocols were validated through simulations, phantom, and in vivo experiments. Ten healthy subjects were recruited to provide sufficient data for the unrolled network. The entire pipeline was validated on additional 5 healthy subjects where different EPTI sampling approaches were compared. Two additional pediatric patients with epilepsy were recruited to demonstrate the generalizability of the unrolled reconstruction.

Results

sEPTI achieved 1.4 × faster imaging with improved image quality and quantitative map precision compared to existing EPTI approaches. The B0 update and the phase correction provide improved reconstruction performance with lower artifacts. The unrolled network boosted the SNR, achieving high-quality T2* and QSM quantification with single average data. High-quality reconstruction was also obtained in the pediatric patient using this network.

Conclusion

sEPTI achieved whole-brain distortion-free multi-echo imaging and T2* and QSM quantification at 0.75 mm in 90 seconds which has the potential to be useful for wide clinical applications.
Discovery and Repurposing of Pharmaceutical Agents as Antifungals:In vivoactivity againstCoccidioides
Authors: Mead Heather L. , Valentine Michael , Yin Holly , Thompson George R. , Keim Paul , Engelthaler David M. , Barker Bridget M.
Published: February 2024

There is significant interest in identifying improved treatments for coccidioidomycosis, an endemic fungal infection found in the southwestern United States, and Central and South America. The current standard of care, fluconazole, often fails to completely eradicateCoccidioidesinfection; however, the cost of identifying new compounds is often high in terms of both finances and time. Therefore, repurposing existing pharmaceutical agents is an attractive option. In our previous work, we identified several compounds which inhibited fungal growthin vitro. Based on these findings, we screened a subset of these agents to increase the performance of fluconazole in a combination therapy approach, as compared to fluconazole alone, in a murine model. We observed that combination therapy of sertraline:fluconazole significantly reduced the amount of live fungus in the lung compared to fluconazole alone. These results suggest that sertraline can be repurposed as an adjunctive agent in the treatment of this important fungal disease.
Investigating the effects of repetitive paired-pulse transcranial magnetic stimulation on visuomotor training using TMS-EEG
Authors: Sasaki Ryoki , Hand Brodie J. , Liao Wei-Yeh , Semmler John G. , Opie George M.
Published: February 2024

Objectives

I-wave periodicity repetitive paired-pulse transcranial magnetic stimulation (iTMS) can modify acquisition of a novel motor skill, but the associated neurophysiological effects remain unclear. The current study therefore used combined TMS-electroencephalography (TMS-EEG) to investigate the neurophysiological effects of iTMS on subsequent visuomotor training (VT).

Methods

Sixteen young adults (26.1 ± 5.1 years) participated in three sessions including real iTMS and VT (iTMS + VT), control iTMS and VT (iTMSsham+ VT), or iTMS alone. Motor-evoked potentials (MEPs) and TMS-evoked potentials (TEPs) were measured before and after iTMS, and again after VT, to assess neuroplastic changes.

Results

Irrespective of the intervention, MEP amplitude was not changed after iTMS or VT (P= 0.211). Motor skill was improved compared with baseline (P< 0.001), but no differences were found between stimulus conditions. In contrast, the P30 peak was altered by VT when preceded by sham iTMS (P< 0.05), but this effect was not apparent when VT was preceded by iTMS or following iTMS alone (allP> 0.15).

Conclusion

In contrast to expectations, iTMS was unable to modulate MEP amplitude or influence motor learning. Despite this, changes in P30 amplitude suggested that motor learning was associated with altered cortical reactivity. Furthermore, this effect was abolished by priming with iTMS, suggesting an influence of priming that failed to impact learning.

Authorship statements

Conceptualization: JGS; Data curation: RS, BJH, and WL; Formal analysis: RS; Funding acquisition: RS; Investigation: RS, BJH, and WL; Methodology: RS, GMO, BJH and JGS; Project administration: GMO and JGS; Supervision: GMO and JGS; Roles/Writing - original draft: RS and GMO; Writing - review & editing: BJH, WL, and JGS.
Convergent evolution of plant prickles is driven by repeated gene co-option over deep time
Authors: Satterlee James W. , Alonso David , Gramazio Pietro , Jenike Katharine M. , He Jia , Arrones Andrea , Villanueva Gloria , Plazas Mariola , Ramakrishnan Srividya , Benoit Matthias , Gentile Iacopo , Hendelman Anat , Shohat Hagai , Fitzgerald Blaine , Robitaille Gina M. , Green Yumi , Swartwood Kerry , Passalacqua Michael J. , Gagnon Edeline , Hilgenhof Rebecca , Huggins Trevis D. , Eizenga Georgia C. , Gur Amit , Rutten Twan , Stein Nils , Yao Shengrui , Bellot Clement , Bendahmane Mohammed , Frary Amy , Knapp Sandra , Särkinen Tiina , Gillis Jesse , Van Eck Joyce , Schatz Michael C. , Eshed Yuval , Prohens Jaime , Vilanova Santiago , Lippman Zachary B.
Published: February 2024

An enduring question in evolutionary biology concerns the degree to which episodes of convergent trait evolution depend on the same genetic programs, particularly over long timescales. Here we genetically dissected repeated origins and losses of prickles, sharp epidermal projections, that convergently evolved in numerous plant lineages. Mutations in a cytokinin hormone biosynthetic gene caused at least 16 independent losses of prickles in eggplants and wild relatives in the genusSolanum. Strikingly, homologs promote prickle formation across angiosperms that collectively diverged over 150 million years ago. By developing newSolanumgenetic systems, we leveraged this discovery to eliminate prickles in a wild species and an indigenously foraged berry. Our findings implicate a shared hormone-activation genetic program underlying evolutionarily widespread and recurrent instances of plant morphological innovation.
Multi-tissue transition of A-to-I RNA editing pattern and its regulatory relevance in transcription, splicing, and translation during development
Authors: Pan Jia-Qi , Pan Xu-Bin , Liu Yan-Shan , Jin Yun-Yun , Chen Jian-Huan
Published: February 2024

Previous studies have shown that A-to-I RNA editing can occur in various organs and tissues of normal physiological conditions. However, the dynamics of RNA editing and its functional relevance in multiple tissues and organs during the embryo-to-adult transition in mammals remains to be elucidated. Herein, we performed a comprehensive analysis of RNA sequencing and ribosome profiling of six mouse tissues at embryonic and adult stages, to elucidate the tissue- and stage-specific landscape of A-to-I RNA editing. Our result identified transcriptome-wide A-to-I RNA editing in six tissue types. Furthermore, differential expression was concurrently observed in a set of distinct differential RNA editing genes at both mRNA and protein levels across different tissues. Gene function and pathway enrichment analysis indicated that these genes with both differential editing and expression were involved in not only tissue-specific biological functions, but also common fundamental processes of post-transcriptional and post-translational modification. Further analysis showed a dynamic interaction between A-to-I RNA editing and alternative splicing in cell survival, death, signal transduction, and cell-cell interactions during development. Overall, our study demonstrates the potential role played by A-to-I editing during development, providing new insight into the effects of RNA editing within both transcriptional and translational landscape on it.
Social touch shapes communication and animal recognition in naked mole-rats
Authors: Schwark Ryan , Ogundare Simon , Sheng Preston , Foster William , Chang Phalaen , Tsai Yu-Young , Arnold Amanda , Abdus-Saboor Ishmail
Published: February 2024

The East African naked mole-rat (Heterocephalus glaber) lives in cooperative subterranean colonies and displays a capacity to recognize social novelty, rank, and identity. The sensory cues used for social recognition remain poorly understood, especially because many of their senses are either lost or greatly reduced in comparison to other mammals. Here, we found that naked mole-rats actively touch faces 100% of the time they encounter one another in a tunnel test, followed by milliseconds speed determination of the rank of the other animal. Even in an open arena, naked mole-rats engage in face-to-face touch hundreds of times in a 10-minute social pairing and colonies do so tens of thousands of times over a 24-hour period in their home environment. To demonstrate the prominence of face touch at a molecular level, we show that social housing conditions lead to widespread activation of mechanosensory ion channels, including Piezo2, in neurons that innervate the face, but not the body. Lastly, to determine the ethological relevance of face touch, we reduced its capacity with facial whisker trimming and revealed an apparent inability for animals to recognize colony members. Together, these findings uncover face touch as a prominent social behavior in naked mole-rats that is intimately linked to social recognition.
A modular toolkit for environmentalRhodococcus, Gordonia, andNocardiaenables complex metabolic manipulation
Authors: Jansen Zachary , Alameri Abdulaziz , Wei Qiyao , Kulhanek Devon L. , Gilmour Andrew R. , Halper Sean , Schwalm Nathan D. , Thyer Ross
Published: February 2024

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome, but largely lack genetic tools comparable to those available in model species such asE. coliorPseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolateRhodococcus ruberC208 and demonstrate conserved functionality in diverse environmental isolates ofRhodococcus, NocardiaandGordonia. This toolkit encompasses Mycobacteriale origins of replication, broad-host range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway inRhodococcus erythropolisN9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux towards the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.

IMPORTANCE

Soil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens.Rhodococcus, GordoniaandNocardiaare three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these,Rhodococcus ruberC208 was shown to degrade polyethylene,Gordonia paraffinivoranscan assimilate carbon from solid hydrocarbons, andNocardia neocaledoniensis(and many otherNocardia) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.
Endovascular thrombectomy with and without preceding thrombolysis in posterior circulation stroke – insights from STAR
Authors: Heide Ev-Christin , Al Kasab Sami , Alawieh Ali , Arthur Adam , Brinjikji Waleed , Chowdhry Shakeel , Crosa Roberto , Cuellar Hugo , De Leacy Reade , Dumont Travis , Ernst Marielle , Ezzeldin Mohamad , Fragata Isabel , Howard Brian , Jabbour Pascal , Kan Peter , Kim Joon-Tae , Levitt Michael R. , Mascitelli Justin , Matouk Charles , Moss Mark , Navia Pedro , Osbun Joshua , Park Min S. , Polifka Adam , Psychogios Marios-Nikos , Rai Ansaar , Romano Daniele G. , Shaban Amir , Starke Robert M. , Tanweer Omar , Williamson Richard , Wolfe Stacey Q. , Yoshimura Shinichi , Spiotta Alejandro M. , Maier Ilko L.
Published: February 2024

Background

Multiple randomized trials could not establish the non-inferiority of endovascular thrombectomy (EVT) alone without preceding intravenous thrombolysis (IVT) or superiority of IVT followed by EVT in anterior circulation large vessel occlusion (LVO) stroke. The role of prior IVT in posterior circulation LVO remains controversial.

Methods

In this multicenter, retrospective study, stroke patients with LVO in the posterior circulation who received EVT alone or with IVT were selected from the stroke thrombectomy and aneurysm registry (STAR) between 2013 to 2022. Effects of IVT followed by thrombectomy on favorable functional outcome (defined as modified Rankin scale ≤ 3 at 90 days) and safety were investigated using multivariable logistic and linear regression models.

Results

Of the 588 included patients, 67 % (n = 394) were treated with EVT alone and 29% (n = 170) with EVT after IVT and 4% (n = 24) have missing values on this variable. Controlling for multiple confounding factors, IVT was not associated with a higher likelihood of favorable functional outcome at 90 days (odds ratio 1.04, 95 % CI 0.52-2.09, p = 0.901). Thrombectomy alone did not show any safety advantages compared with those receiving IVT.

Conclusions

Similar functional outcomes and complication rates were seen in patients with posterior circulation LVO treated with EVT alone vs EVT after IVT. Further prospective studies are required to determine the utility of IVT in posterior circulation stroke, especially in patients being directly admitted to thrombectomy centers.
Time Resolved Inspection of Ionizable-Lipid Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Endosomal Membrane Mimic
Authors: Aliakbarinodehi Nima , Niederkofler Simon , Olsén Erik , Jing Yujia , Emilsson Gustav , Sjöberg Mattias , Agnarsson Björn , Lindfors Lennart , Höök Fredrik
Published: February 2024

This study investigates pH-triggered fusion dynamics of lipid nanoparticles (LNPs) with an endosomal membrane mimic, addressing mechanistic aspects of a crucial yet elusive process for effective mRNA delivery. Utilizing time-resolved total internal reflection fluorescence (TIRF) imaging, we observed a delayed onset of LNP fusion upon pH drop, lasting seconds to minutes depending on pH and LNP size. Once initiated, LNP fusion and cargo escape occurred rapidly within tens to hundreds of milliseconds. While LNP disintegration is observed to lead to release of a significant portion of mRNA into the acidic environment, some mRNA molecules remained mobile on the endosomal membrane mimic due to deprotonation-resistant complex salt formation. Comparison of the fusion efficiency of two LNP formulations correlated with protein translation in human primary cell transfection data, emphasizing the importance of biophysical investigations in understanding ionizable-lipid-containing LNP-assisted mRNA delivery mechanisms and providing insights for optimizing mRNA-LNP design for enhanced endosomal escape.
Capturing and Characterising Wild Yeast for Beer Brewing
Authors: Kerr Edward D. , Howes Mark T. , Schulz Benjamin L.
Published: February 2024

Beer is typically made using fermentation withSaccharomyces cerevisiaeorSaccharomyces pastorianus, domesticated brewing yeasts. Historically, wild, non-Saccharomycesyeasts have also been frequently used in mixed culture fermentations to provide interesting and unique flavours to beer. However, brewing using mixed cultures or by spontaneous fermentation makes reproducing flavours and beer styles extremely difficult. Here, we describe a pipeline from collection of wild yeast from plant material to the characterisation and industrial scale production of beer using wild yeast. We isolated and identified wild yeast strains from the St Lucia campus of The University of Queensland, Brisbane, Australia. Several isolates fermented efficiently at laboratory scale, but failed to grow at industrial scale due to the combination of maltose and pressure stress. Systems biology showed that the synergistic metabolic defects caused by these dual stresses converged on amino acid nutrient uptake. Glucoamylase addition relieved maltose stress and allowed industrial scale fermentation using wild yeast. Our workflow allows efficient collection and characterisation of diverse wild yeast isolates, identification of interventions to allow their use at industrial scale, and investigation of the genetic and metabolic diversity of wild yeasts.
Cre-Lox miRNA-delivery technology optimized for inducible microRNA and gene-silencing studies in zebrafish
Authors: Guo Fangfei , Tromp Alisha , Wang Haitao , Hall Thomas E , Giacomotto Jean
Published: February 2024

While many genetic tools exist for zebrafish, this animal model is still lacking a robust gene-silencing and microRNA-delivery technology enabling spatio-temporal control and reliable traceability. We have recently demonstrated that the use of engineered pri-miR backbones can be used to both trigger stable and heritable gene knockdown and/or express microRNA(s) of choice in this organism. This approach/material fills a gap that exists in the zebrafish genetic toolbox to study microRNA biology and has the potential to be a powerful complementary tool to CRISPR-knockout and CRISPR-interference approaches by enabling conditional, traceable tissue-specific and polygenic gene-silencing. However, to date, this miRNA-expressing technology still presents important limitations. First, to trigger potent knockdown(s), multiple synthetic-miRNAs must be expressed simultaneously which compromises the co-expression of fluorescent marker(s) and knockdown traceability, thereby reducing the interest and versatility of this approach. Second, when the gene knockdown triggers significant phenotypes, like homozygous mutants with severe early phenotypes, it is difficult, if not impossible, to maintain adult transgenic carriers. Here, to solve these problems and provide a mature RNAi and microRNA-delivery technology for zebrafish research, we have generated and validated new RNAi reagents and an inducible delivery system based on the Cre/Lox technology. This new system allows the creation of asymptomatic/silent carriers, easing the production of embryos with potent knockdowns that can be traced and spatiotemporally controlled. Following this technological development, we demonstrate the utility of our approach by generating novel inducible models of spinal muscular atrophy (SMA) that open new avenues for studying the normal and pathogenic functions of the smn1 gene, as well as for establishing large-scale screens. Finally, and very importantly, the materials and techniques developed here will simplify research into microRNA biology, as well as conditions involving haploinsufficiency and multiple genes.
Heterozygosity for Crohn’s Disease Risk Allele ofATG16L1Protects against Bacterial Infection
Authors: Yao Xiaomin , Rudensky Eugene , Martin Patricia K. , Zwack Erin E. , He Zhengxiang , Furtado Glaucia C. , Lira Sérgio A. , Torres Victor J. , Shopsin Bo , Cadwell Ken
Published: February 2024

The T300A substitution in ATG16L1 associated with Crohn’s Disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1T316A), but not homozygosity, protects against lethalSalmonella entericaTyphimurium infection. One copy ofAtg16L1T316Awas sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies ofAtg16L1T316Ainhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous forATG16L1T300Adisplayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence.

One-Sentence Summary

Heterozygosity of Crohn’s diseases risk variantATG16L1 T300Aconfers protection against bacterial infections.
A comprehensive personal omics clinical interpreter based on genomic and transcriptomic profiles
Authors: Liu Yaqing , Chen Qingwang , Chen Qiaochu , Sang Leqing , Wang Yunjin , Shi Leming , Zheng Yuanting , Guo Li , Yu Ying
Published: February 2024

Advances in precision medicine rely on the accurate identification and analysis of molecular alterations for personalized diagnostic, prognostic, and therapeutic decision-making. A critical obstacle is the integration of heterogeneous interpretations of clinically actionable alterations from various knowledgebases. Here, we present the Personal Omics Interpreter (POI), a web-based application engineered to aggregate and interpret therapeutic options, including targeted, immunological, and chemotherapeutic agents, by leveraging personal genomic and transcriptomic profiles. POI employs the Precision Medicine Knowledgebase (PreMedKB), an updated harmonized resource we previously reported, to annotate the clinically actionable somatic variants. It further incorporates a predictive algorithm to broaden therapeutic options according to established gene-gene interactions and offers insights into phenotypic responses of chemotherapeutic agents through phasing germline diplotypes. Validated against three cohort datasets encompassing over 22,000 cancer patients, POI demonstrates consistently high matching rates (94.7 ∼ 95.6%) between patients and suggested therapies, highlighting its potential in supporting precision-driven informed treatment strategies.
Criterion placement threatens the construct validity of neural measures of consciousness
Authors: Fahrenfort Johannes J. , Johnson Philippa A. , Kloosterman Niels A. , Stein Timo , van Gaal Simon
Published: February 2024

How consciousness arises from brain activity has been a topic of intense scientific research for decades. But how does one identify the neural basis of something that is intrinsically personal and subjective? A hallmark approach has been to ask observers to judge stimuli as ‘seen’ (conscious) and ‘unseen’ (unconscious) and use post hoc sorting of neural measurements based these judgments. Unfortunately, cognitive and response biases are known to strongly affect how observers place their criterion for judging stimuli as ‘seen’ vs. ‘unseen’, thereby confounding neural measures of consciousness. Surprisingly however, the effect of conservative and liberal criterion placement on neural measures of unconscious and conscious processing has never been explicitly investigated. Here we use simulations and electrophysiological brain measurements to show that conservative criterion placement has an unintuitive consequence: rather than selectively providing a cautious estimate of conscious processing, it inflates effect sizes in neural measures of both conscious and unconscious processing, while liberal criterion placement does the reverse. After showing this in simulation, we performed decoding analyses on two electroencephalography studies that employ common subjective indicators of conscious awareness, in which we experimentally manipulated the response criterion. The results confirm that the predicted confounding effects of criterion placement on neural measures of unconscious and conscious processing occur in empirical data, while further showing that the most widely used subjective scale, the Perceptual Awareness Scale (PAS), does not guard against criterion confounds. Follow-up simulations explicate how the experimental context determines whether the relative confounding effect of criterion placement is larger in neural measures of either conscious or unconscious processing. We conclude that criterion placement threatens the construct validity of neural measures of conscious and unconscious processing.
Rapid, direct, and sequence-specific identification of RNA viruses in various crop plants using CRISPR/Cas13a
Authors: Hak Hagit , Ostendorp Steffen , Reza Anton , Ishgur Greenberg Shany , Pines Gur , Kehr Julia , Spiegelman Ziv
Published: February 2024

Plant viruses are destructive pathogens causing significant damage to various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a useful tool for plant virus identification. However, its application for on-site, direct detection of viruses from plant tissues is still limited. In this study, we present a rapid method for detecting viruses directly from RNA of different crop species using CRISPR/Cas13a. We successfully applied this method to identify tomato brown rugose fruit virus (ToBRFV) in infected tomato plants and differentiate it from closely related tobamoviruses. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Moreover, CRISPR/Cas13a was used to directly identify cucumber green mottle mosaic virus (CGMMV) in cucumber plants and turnip mosaic virus (TuMV) inBrassica napusplants. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site ToBRFV identification using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV detection in a commercial greenhouse. These results demonstrate that CRISPR/Cas13a is a robust technology for direct, rapid, sensitive, and specific identification of multiple viruses in different crop plants that can be easily implemented for on-site detection.
Trends in genome diversity of small populations under a conservation program: a case study of two French chicken breeds
Authors: Bortoluzzi Chiara , Restoux Gwendal , Rouger Romuald , Desnoues Benoit , Petitjean Florence , Bosse Mirte , Tixier-Boichard Michèle
Published: February 2024

Livestock biodiversity is declining globally at rates unprecedented in human history. Of all avian species, chickens are among the most affected ones because many local breeds have a small effective population size that makes them more susceptible to demographic and genetic stochasticity. The maintenance of genetic diversity and control over genetic drift and inbreeding by conservation programs are fundamental to ensure the long-term survival and adaptive potential of a breed. However, while the benefits of a conservation program are well understood, they are often overlooked. We here used temporal whole-genome sequencing data to assess the effects of a conservation program on the genetic diversity (Δπ), deleterious variation (ΔL), and inbreeding (ΔF) of two local French chicken breeds, the Barbezieux and Gasconne. We showed that when the conservation program is consistent over time and does not undergo any major organisational changes (i.e., Barbezieux), the loss of genetic diversity is limited. This was true for both pedigree and genomic inbreeding but also for the genetic load estimated from functionally important genome-wide variants. However, when a conservation program is interrupted or re-initiated from scratch (i.e., Gasconne), the loss of genetic diversity can hardly be limited as a result of the bottleneck effect associated with the re-sampling. Our results reinforce the imperative to establish and sustain existing conservation programs that aim to keep populations with a relatively small effective population size from the brink of extinction. Moreover, we conclude by encouraging the use of molecular data to more effectively monitor inbreeding at the genome level while improving fitness by tracking protein-coding and non-coding deleterious variants.
Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis
Authors: Chougoni Kranthi Kumar , Neely Victoria , Ding Boxiao , Oduah Eziafa , Lam Vianna , Hu Bin , Koblinski Jennifer E. , Windle Bradford E. , Deb Swati Palit , Deb Sumitra , Radhakrishnan Senthil K. , Harada Hisashi , Grossman Steven R.
Published: February 2024

Non-small cell lung cancer (NSCLC) cells with oncogenic mutant p53 alleles (Onc-p53) exhibit significantly higher levels of proteasome activity, indicating that Onc-p53 induces proteotoxic stress which may be leveraged as a therapeutic vulnerability. Proteasome inhibitors (PIs), such as bortezomib (BTZ), can induce toxic levels of oxidative stress in cancer cells and thus we investigated whether PIs exhibit preferential cytotoxicity in Onc-p53 NSCLC cells. Indeed, BTZ and other PIs exhibited the IC506-7-fold lower in Onc-p53 cells vs. wild-type (WT) p53 cells. BTZ cytotoxic effects in Onc-p53 cells were nearly completely rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ-dependent cytotoxic effects in Onc-p53 cells. Importantly, we observed oxidative stress-dependent transcriptional induction of the pro-apoptotic NOXA with downstream cleaved caspase-3, consistent with apoptotic cell death in Onc-p53 but not in WT p53 cells treated with BTZ, and BTZ-generated oxidative stress was linked to nuclear translocation of NRF2 and transcriptional activation of ATF3, which in turn was required for NOXA induction. Validating BTZ’s translational potential in Onc-p53 NSCLC, BTZ and carboplatin or the BH3-mimetic navitoclax were synergistically cytotoxic in Onc-p53 but not WT p53 cellsin vitro,and BTZ effectively limited growth of Onc-p53 NSCLC xenografts when combined with either carboplatin or navitoclaxin vivo. Our data therefore support further investigation of the therapeutic utility of PIs combined with carboplatin or BH3-mimetics in Onc-p53 human NSCLC as novel therapeutic strategies.

Significance

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death due, in part, to a lack of active therapies in advanced disease. We demonstrate that proteasome inhibitor/BH3-mimetic combination therapy is an active precision therapy in NSCLC cells and tumors expressing oncogenic mutant p53 alleles (Onc-p53).