bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell specificity of adeno-associated virus (AAV) serotypes in human cortical organoids
Authors: Stanton Morgan M. , Hariani Harsh N. , Sorokin Jordan , Taylor Patrick M. , Modan Sara , Rash Brian G. , Rao Sneha B. , Enriquez Luigi , Quang Daphne , Hsu Pei-Ken , Paek Justin , Owango Dorah , Castrillo Carlos , Nicola Justin , Ramkumar Pavan , Lash Andy , Flanzer Douglas , Shah Kevan , Kato Saul , Skibinski Gaia
Published: April 2023

Human-derived cortical organoids (hCOs) recapitulate cell diversity and 3D structure found in the human brain and offer a promising model for discovery of new gene therapies targeting neurological disorders. Adeno-associated viruses (AAVs) are the most promising vehicles for non-invasive gene delivery to the central nervous system (CNS), but reliable and reproduciblein vitromodels to assess their clinical potential are lacking. hCOs can take on these issues as they are a physiologically relevant model to assess AAV transduction efficiency, cellular tropism, and biodistribution within the tissue parenchyma, all of which could significantly modulate therapeutic efficacy. Here, we examine a variety of naturally occurring AAV serotypes and measure their ability to transduce neurons and glia in hCOs from multiple donors. We demonstrate cell tropism driven by AAV serotype and hCO donor and quantify fractions of neurons and astrocytes transduced with GFP as well as overall hCO health.
Single-Cell Transcriptomes and Immune Repertoires Reveal the Cell State and Molecular Changes in Pemphigus Vulgaris
Authors: Duan Shumin , Li Qionghua , Wang Fei , Kuang Wenjing , Dong Yunmei , Liu Dan , Wang Jiongke , Li Wei , Chen Qianming , Zeng Xin , Li Taiwen
Published: April 2023

The etiology and pathogenesis of pemphigus vulgaris (PV) are closely related to both immune cells and epithelial cells, but the specific subtypes of immune cells involved in PV and their roles are not yet fully understood. Additionally, the specific functions and mechanisms of first-line treatment glucocorticoids on cell types of PV remain to be elucidated. We performing 5’ single-cell RNA sequencing, combined with V(D)J enrichment on buccal mucosal lesions and peripheral blood samples from treatment-naïve patients with PV, in conjunction with post-treatment peripheral blood samples obtained after oral prednisone treatment. Our findings suggest that IL-1α signaling pathway, myeloid antigen presenting cells, inflammatory CD8+ Trm, and dysfunctional CD4+ Treg are crucial in PV. Our results were also supported by immunohistochemical assays. Furthermore, our results show that prednisone has a significant impact on monocytes and MAIT, but a limited effect on CD4+ Treg. Finally, we provide CDR3 amino acid sequence data of BCR that may be used as therapeutic targets. In conclusion, this study provides a comprehensive understanding of PV, particularly in the mucosal-dominant type, and the effect of GCs on PV, which could effectively lead to the development of new therapeutic strategies.
Parting the cellular sea: electrotaxis evoked directional separation of co-cultured keratinocytes and fibroblasts
Authors: Leal José , Shaner Sebastian , Jedrusik Nicole , Savelyeva Anna , Asplund Maria
Published: April 2023

Bioelectric communication plays a significant role in several cellular processes and biological mechanisms, such as division, differentiation, migration, cancer metastasis, or wound healing. The flow of ions through cellular walls and the gradients generated thereby evoke this signaling as electric fields (EFs) form across membranes, and their changes act as cues for cells. An EF is natively generated towards the wound center during epithelial wound healing, aiming to align and guide cell migration, particularly of keratinocytes, fibroblasts, and macrophages. While this phenomenon, known as electrotaxis, has been extensively investigated across many cell types, it is typically explored one cell type at a time, which does not accurately represent cellular interactions during complex biological processes. Here we show the co-cultured electrotaxis of epithelial keratinocytes and fibroblasts with a salt-bridgeless microfluidic approach for the first time. The electrotactic response of these cells was first assessed in mono-culture to establish a baseline, resulting in a characteristic anodic migration for keratinocytes and cathodic for fibroblasts. Both cell types retained their electrotactic properties in co-culture leading to clear cellular partition. The methods leveraged herein can pave the way for future co-culture electrotaxis experiments where the concurrent influence of cell lines can be thoroughly investigated.
Uptake without inactivation of human adenovirus type 2 byTetrahymena pyriformisciliates
Authors: Olive Margot , Daraspe Jean , Genoud Christel , Kohn Tamar
Published: April 2023

Human adenoviruses are ubiquitous contaminants of surface water. Indigenous protists may interact with adenoviruses and contribute to their removal from the water column, though the associated kinetics and mechanisms differ between protist species. In this work, we investigated the interaction of human adenovirus type 2 (HAdV2) with the ciliateTetrahymena pyriformis. In co-incubation experiments in a freshwater matrix,T. pyriformiswas found to efficiently remove HAdV2, with ≥ 4 log10removal over 72 hours. Neither sorption onto the ciliate nor secreted compounds contributed to the observed loss of infectious HAdV2. Instead, internalization was shown to be the dominant removal mechanism, resulting in the presence of viral particles inside food vacuoles ofT. pyriformis,as visualized by transmission electron microscopy. The fate of HAdV2 once ingested was scrutinized and no evidence of virus digestion was found over the course of 48 hours. This work shows thatT. pyriformiscan exert a dual role on microbial water quality: while they remove infectious adenovirus from the water column, they can also accumulate and potentially protect infectious viruses from exposure to environmental or engineered stressors.

Environmental significance

Human viruses are ubiquitous contaminants of surface water. The fate of human viruses, once discharged into the environment, is modulated by indigenous microorganisms in the surrounding water body. Among microorganisms, the role of protists in controlling virus persistence has been overlooked. Here, we investigate the interactions of the ciliateTetrahymena pyriformiswith human adenovirus type 2. We demonstrate thatT. pyriformiscan serve as a sink of adenovirus from the water column, but also act as a reservoir and potential protective barrier for infectious viruses against stressors.T. pyriformisciliates are among the most abundant protists in surface waters. Understanding this dual role of protists is important for assessing and maintaining microbial water quality and infection risks.
A novel high-throughput assay identifies small molecules with activity against persister cells
Authors: Petersen Maiken Engelbrecht , Hansen Liva Kjær , Kelly Nicholas M. , Wood Thomas Keith , Jørgensen Nis Pedersen , Østergaard Lars Jørgen , Meyer Rikke Louise
Published: April 2023

Persister cells are a subpopulation of transiently antibiotic tolerant bacteria, which are believed to be the main cause of relapsing bacterial infections. Due to the importance of persister cells in human infections, there is a need for new antibiotics that kill bacteria independently of their activity. However, high-throughput assays to screen for drugs with such activity are missing. This is partly due to the transient nature of the phenotype, which makes it is difficult to prepare a concentrated population of persister cells that remain inactive during incubation with antibiotics in standard growth media.

The purpose of this study was to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells during a 24 h incubation period. Subsequently, this assay was used to screen a selection of small molecules with hypothesized antimicrobial activity.

The fraction ofS. aureusthat tolerate bactericidal concentrations of ciprofloxacin were defined as persister cells. We first quantified how the cell concentration, growth phase, antibiotic concentration, duration of antibiotic exposure, and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we compared our approach to generate persister cells, to a process that generated persister cells by a short exposure to rifampicin. Finally, we used the optimised protocol to identify molecular structures that have anti-persister activity by performing screening on initially compound fragments and then selecting compounds that incorporated the fragments that displayed activity.

We show that exponential- and stationary-phase cultures transferred to nutrient-rich media only contain a small fraction (0.001 to 0.07 %) of persister cells that tolerated 10, 50 and 100 × MIC ciprofloxacin. Exponential-phase cultures displayed a bi-phasic time-kill curve, which plateaued after 5 h exposure, while stationary phase cultures displayed a low, but constant death rate at 50 and 100 × MIC ciprofloxacin. Inducing the persister phenotype with a short rifampicin treatment resulted in 100% persister cells when evaluated after ≤5 h exposure to ciprofloxacin. However, after longer incubation times, cells resumed activity and lost their tolerance to ciprofloxacin. Tolerance was only maintained in the majority of the population for the full 24 h incubation period if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. We conclude that keeping cells starved in a carbon-free medium enables generation of high concentrations ofS. aureuscells that tolerate 50 × MIC ciprofloxacin, and we find this protocol easily applicable for rapid screening of anti-persister drugs that act on dormant or non-dividing cells.
Virus-induced overexpression of heterologous FT for efficient speed breeding in tomato
Authors: Deng Yingtian , Yarur-Thys Antonia , Baulcombe David
Published: April 2023

Potato Virus X (PVX) vectors expressing theArabidopsis thaliana FTor tomatoFTorthologSINGLE-FLOWER TRUSS(SFT) accelerated tomato flowering time by 14-21d and increased by 2-3 fold the number of flowers and ripe fruit compared with non-infected or empty vector-infected plants. TheArabidopsisFT was more effective than the tomato orthologue and there was no alteration of the plant architecture, leaf, flower, or fruit morphology. The virus is not transmitted to the next generation and viral vectors with expression of a heterologous FT will be a useful approach to speed breeding in tomato and other species.
GluK2 is a target for gene therapy in drug-resistant Temporal Lobe Epilepsy
Authors: Boileau Céline , Deforges Severine , Peret Angélique , Scavarda Didier , Bartolomei Fabrice , Giles April , Partouche Nicolas , Gautron Justine , Viotti Julio , Janowitz Haley , Penchet Guillaume , Marchal Cécile , Lagarde Stanislas , Trebuchon Agnès , Villeneuve Nathalie , Rumi Julie , Marissal Thomas , Khazipov Roustem , Khalilov Ilgam , Martineau Fanny , Maréchal Marine , Lepine Anne , Milh Mathieu , Figarella-Branger Dominique , Dougy Etienne , Tong Soutsakhone , Appay Romain , Baudouin Stéphane , Mercer Andrew , Smith Jared B. , Danos Olivier , Porter Richard , Mulle Christophe , Crépel Valérie
Published: April 2023

Objective

Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) creates an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence there is an urgent need for novel therapies. Previously we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE.

Methods

We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE.

Results

Here we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated Interictal-like Epileptiform Discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2miRNA was designed to specifically downregulate GluK2 expression. Direct delivery of AAV9-antigrik2miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs.

Interpretation

Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients.
Design of a stem cell-based therapy for ependymal repair in hydrocephalus associated with germinal matrix hemorrhages
Authors: Rodríguez-Pérez Luis M. , Ojeda-Pérez Betsaida , García-Bonilla María , López-de San Sebastián Javier , González-García Marcos , Fernández-Muñoz Beatriz , Sánchez-Pernaute Rosario , García-Martín María L. , Domínguez-Pinos Dolores , Cárdenas-García Casimiro , Jiménez Antonio J. , Páez-González Patricia
Published: April 2023

Germinal matrix hemorrhages (GMH) and the consequent posthemorrhagic hydrocephalus (PHH) are among the most common and severe neurological complications of preterm birth that require lifelong complex neurosurgical care. GMH and PHH provoke disruption of neuroepithelium/ependyma development, a key structure implicated in brain development and homeostasis. Neuroepithelial/ependymal damage causes lifelong cognitive and motor deficits; however, no therapy is directed to recover the damaged ependyma. This study is aimed to test the possibilities of ependymal repair in GMH/PHH using neural stem cells (NSCs) or ependymal progenitors (EpPs). Thus, it sets the basis for a therapeutic approach to treating ependymal damage and preventing brain developmental deficits. GMH/PHH was induced in 4-day-old mice using different experimental procedures involving collagenase, blood, or blood serum injections. PHH severity was characterized using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Additionally, a newexvivoapproach using ventricular walls from mice developing moderate and severe GMH/PHH was generated to study ependymal restoration and wall regeneration after stem cell treatments. NSCs or EpPs obtained from newborn mice were transplanted in the explants, and pretreatment with mesenchymal stem cells (MSCs) was tested. Ependymal differentiation and the effect of MSC-conditioned microenvironment were investigated in both explants and primary cultures. In the animals, PHH severity was correlated with the extension of GMH, ependymal disruption, astroglial/microglial reactions, and ventriculomegaly. In the explants, the severity and extension of GMH hindered the survival rates of the transplanted NSCs/EpPs. In the explants affected with GMH, new multiciliated ependymal cells could be generated from transplanted NSCs and, more efficiently, from EpPs. Blood and TNFα negatively affected ciliogenesis in cells expressing Foxj1. Pretreatment with mesenchymal stem cells (MSC) improved the survival rates of EpPs and ependymal differentiation while reducing the edematous and inflammatory conditions in the explants. In conclusion, in GMH/PHH, the ependyma can be restored from either NSC or EpP transplantation, being EpPs in an MSC-conditioned microenvironment more efficient for this purpose. Modifying the neuroinflammatory microenvironment by MSC pretreatment positively influenced the success of the ependymal restoration.
Mouse gingival single-cell transcriptomic atlas: An activated fibroblast subpopulation guides oral barrier immunity in periodontitis
Authors: Kondo Takeru , Gleason Annie , Okawa Hiroko , Hokugo Akishige , Nishimura Ichiro
Published: April 2023

Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulationactivated toguide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils together with AG fibroblasts appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis ofRag2-/- andRag2γc-/-mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose that AG fibroblasts function as a previously unrecognized surveillant to initiate gingival inflammation leading to periodontitis through the AG fibroblast-neutrophil-ILC3 axis.
MdMYB44-like positively regulates salt and drought tolerance via the MdPYL8-MdPP2CA module in apple
Authors: Chen Cui , Zhang Zhen , Lei Ying-Ying , Chen Wen-Jun , Zhang Zhi-Hong , Li Xiao-Ming , Dai Hong-Yan
Published: April 2023

Abscisic acid (ABA) is involved in salt and drought stress responses, but the underlying molecular mechanism remains unclear. Here, we demonstrated that the overexpression ofMdMYB44-like, an R2R3-MYB transcription factor (TF), significantly increases the salt and drought tolerance of transgenic apple and Arabidopsis. MdMYB44-like inhibits the transcription ofMdPP2CA, which encodes a type 2C protein phosphatase that acts as a negative regulator in ABA response, thereby enhancing ABA signaling-mediated salt and drought tolerance. Furthermore, we found that MdMYB44-like and MdPYL8, an ABA receptor, form a protein complex that further enhances the transcriptional inhibition of theMdPP2CApromoter by MdMYB44-like. Significantly, we discovered that MdPP2CA can interfere with the physical association between MdMYB44-like and MdPYL8 in the presence of ABA, partially blocking the inhibitory effect of the MdMYB44-like-MdPYL8 complex on theMdPP2CApromoter. Thus, MdMYB44-like, MdPYL8, and MdPP2CA form a regulatory loop that tightly controls ABA signaling homeostasis under salt and drought stress. Our data revealed a previously unidentified mechanism by which MdMYB44-like precisely modulates ABA-mediated salt and drought tolerance in apple through the MdPYL8-MdPP2CA module.
Optogenetic ion pumps differ with respect to the secondary pattern of K+redistribution
Authors: Parrish R. Ryley , Jackson-Taylor Tom , Voipio Juha , Trevelyan Andrew J.
Published: April 2023

We recently reported that strong activation of the optogenetic chloride pump, Halorhodopsin leads to a secondary redistribution of K+ions into the cell, through tonically open, “leak” K+channels. Here we show that this effect is not unique to halorhodopsin, but is also seen with activation of another electrogenic ion pump, archaerhodopsin. The two opsins differ however in the size of the rebound rise in extracellular potassium, [K+]o, after the end of activation, which is far larger with halorhodopsin than for archaerhodopsin activation. Multiple linear regression modelling indicates that most of the variance in the post-illumination surge in [K+]o was explained by the type of opsin, and almost nothing by the size of the preceding, illumination-induced drop in [K+]o. These data provide additional support for the hypothesis that intense chloride-loading of cells, as occurs naturally following intense bursts of GABAergic synaptic bombardment, or artificially following halorhodopsin activation, is followed by extrusion of both Cl and K+coupled together. We discuss this with respect to the pattern of [K+]o rise that occurs at the onset of seizure-like events.
Transgenerational Epigenetic Inheritance of MHC Class I Gene Expression is Regulated by the CCAAT Promoter Element
Authors: Weissman Jocelyn D. , Kotekar Aparna , Barbash Zohar , Mu Jie , Singer Dinah S.
Published: April 2023

Transgenerational epigenetic inheritance is defined as the transmission of traits or gene expression patterns across multiple generations that do not derive from DNA alterations. The effect of multiple stress factors or metabolic changes resulting in such inheritance have been documented in plants, worms and flies and mammals. The molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. In this study, we show that mutation of a promoter element, the CCAAT box, disrupts stable expression of an MHC Class I transgene, resulting in variegated expression among progeny for at least 4 generations in multiple independently derived transgenic lines. Histone modifications and RNA polII binding correlate with expression, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box abrogates NF-Y binding and results in changes to CTCF binding and DNA looping patterns across the gene that correlate with expression status from one generation to the next. These studies identify the CCAAT promoter element as a regulator of stable transgenerational epigenetic inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study could provide important insights into how fidelity of gene expression patterns is maintained through multiple generations.
Rapid bacteria-phage coevolution drives the emergence of multi-scale networks
Authors: Borin Joshua M. , Lee Justin J. , Lucia-Sanz Adriana , Gerbino Krista R. , Weitz Joshua S. , Meyer Justin R.
Published: April 2023

Interactions between species have catalyzed the evolution of multiscale ecological networks–including both nested and modular elements that regulate the function of diverse communities. One common assumption is that such complex pattern formation requires long evolutionary timescales, spatial isolation, or other exogenous processes. Here we show that multiscale network structure can evolve rapidly under simple ecological conditions without spatial structure. In just 21 days of laboratory coevolution,Escherichia coliand bacteriophage Φ21 coevolve and diversify to form elaborate cross-infection networks. By measuring ∼10,000 phage–bacteria infections and testing the genetic basis of interactions, we identify the mechanisms that create each component of the multiscale pattern. Initially, nested patterns form through an arms race where hosts successively lose the original receptor (LamB) and phages evolve to use a second (OmpC) and then a third (OmpF) receptor. Next, modules form when the cost of losing the third receptor, OmpF, increases and bacteria evolve resistance mutations that modify the OmpF receptors’ extramembrane loops. In turn, phages evolve adaptations that facilitate specialized interactions with different OmpF variants. Nestedness reemerges within modules as bacteria evolve increased resistance and phages enhance infectivity against module-specific receptor variants. Our results demonstrate how multiscale networks evolve in parasite-host systems, illustrating Darwin’s idea that simple adaptive processes can generateentangled banksof ecological interactions.
Pararectus versus ilioinguinal approach for the management of acetabular fractures: A Protocol for Systemic Review and Meta-analysis
Authors: Rajnish Rajesh Kumar , Srivastava Amit , Yadav Sandeep Kumar , Elhence Abhay , Rathor Kuldeep , Gupta Saurabh
Published: April 2023

Background

Acetabular fractures result from high-energy trauma, and their complex anatomy poses a challenge to surgeons for open reduction and internal fixation of these fractures. The goal of fixation is an anatomical reduction of articular surfaces and stable fixation with minimal damage to vital structures around. The long-term clinical outcomes of these fractures are significantly impacted by selecting an appropriate surgical approach with minimal complications to achieve an anatomical reduction with congruent articular surfaces. The purpose of this review is to compare the outcomes of the pararectus (PR) versus the ilioinguinal (IL) approach for the open reduction and internal fixation (ORIF) of displaced acetabular fractures by looking at the evidence in the existing literature.

Methods

A systematic review and meta-analysis will be performed in accordance with the PRISMA guidelines. A primary search will be done through the electronic databases of PubMed/Medline, Embase, Scopus, and the Cochrane Library using a pre-defined search strategy. A comparative study, either randomized control trials (RCTs) or non-randomized trials, which have compared at least one outcome of the internal fixation of acetabular fractures using the PR versus IL approach will be included in the current review.

Single-armed non-comparative studies, conference posters, abstracts, case reports, review articles, cadaveric studies, book chapters, technical tips, and biomechanical studies will be excluded. Both qualitative and quantitative data analysis will be performed. Appropriate tables and diagrams will be used to demonstrate the qualitative data presentation, and wherever feasible, a quantitative analysis will be done with the appropriate software. The risk-of-bias assessment for non-randomized comparative studies will be done using the MINORS tool, and the Cochrane Collaborations risk-of-bias tool will be used for RCTs.
Mechanisms of regulation of cryptic prophage-encoded gene products inEscherichia coli
Authors: Ragunathan Preethi T. , Lim Evelyne Ng Kwan , Ma Xiangqian , Massé Eric , Vanderpool Carin K.
Published: April 2023

ThedicBFoperon of Qin cryptic prophage inEscherichia coliK12 encodes the small RNA (sRNA) DicF and small protein DicB, which regulate host cell division and are toxic when overexpressed. While new functions of DicB and DicF have been identified in recent years, the mechanisms controlling the expression of thedicBFoperon have remained unclear. Under standard laboratory growth conditions, transcription fromdicBp,the major promoter of thedicBFoperon, is repressed by DicA. Here, we discovered that transcription of thedicBFoperon and processing of the polycistronic mRNA is regulated by multiple mechanisms. DicF sRNA accumulates during stationary phase and is processed from the polycistronicdicBFmRNA by the action of both RNase III and RNase E. DicA-mediated transcriptional repression ofdicBpcan be relieved by an antirepressor protein, Rem, encoded on the Qin prophage. Ectopic production of Rem results in cell filamentation due to strong induction of thedicBFoperon and filamentation is mediated by DicF and DicB. Spontaneous derepression ofdicBpoccurs in a subpopulation of cells independent of the antirepressor. This phenomenon is reminiscent of the bistable switch of λ phage with DicA and DicC performing functions similar to CI and Cro, respectively. Additional experiments demonstrate stress-dependent induction of thedicBFoperon. Collectively, our results illustrate that toxic genes encoded on cryptic prophages are subject to layered mechanisms of control, some that are derived from the ancestral phage and some that are likely later adaptations.

Importance

Cryptic or defective prophages have lost genes necessary to excise from the bacterial chromosome and produce phage progeny. In recent years, studies have found that cryptic prophage gene products influence diverse aspects of bacterial host cell physiology. However, to obtain a complete understanding of the relationship between cryptic prophages and the host bacterium, identification of the environmental, host or prophage-encoded factors that induce the expression of cryptic prophage genes is crucial. In this study, we examine the regulation of a cryptic prophage operon inEscherichia coliencoding a small RNA and a small protein that are involved in inhibiting bacterial cell division, altering host metabolism, and protecting the host bacterium from phage infections.
FOXL2 interaction with different binding partners regulates the dynamics of ovarian development
Authors: Migale Roberta , Neumann Michelle , Mitter Richard , Rafiee Mahmoud-Reza , Wood Sophie , Olsen Jessica , Lovell-Badge Robin
Published: April 2023

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing ofFoxl2deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes suggesting different roles across development. Here, we comprehensively investigated FOXL2’s role through a multi-omics approach to characterise gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in granulosa cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle activation (PFA) and steroidogenesis. Deletion of one interactor, Ubiquitin specific protease 7 (USP7), induces PFA blockage, impaired ovary development and sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.
Event-Induced Modulation of Aperiodic Background EEG: Attention-Dependent and Age-Related Shifts in E:I balance, and Their Consequences for Behavior
Authors: Kałamała Patrycja , Gyurkovics Máté , Bowie Daniel C. , Clements Grace M. , Low Kathy A. , Dolcos Florin , Fabiani Monica , Gratton Gabriele
Published: April 2023

The broadband shape of the EEG spectrum, summarized using a 1/fxfunction, is thought to reflect the balance between excitation and inhibition in cortical regions (E:I balance). This balance is an important characteristic of neural circuits and could inform studies of aging, as older adults show a relative inhibitory activity deficit. Thus far, no studies have leveraged the event-related temporal dynamics of 1/fxactivity to better understand the phases of information processing, especially in the context of aging. Here, for the first time, we examined variations of this activity during the foreperiod of a cued flanker task in younger (YA) and older adults (OA), with picture cues varying in task relevance, relative novelty, and valence. We report a biphasic change in the spectral exponent (corresponding to negative slopes in log-log space) after cue presentation, independent of cue-elicited ERPs, with an initial period of increased negativity (indicating cortical inhibition, similar in YA and OA) followed by decreased negativity (indicating cortical excitation, especially in OA). The decrease in the exponent negativity was associated with lower performance and greater congruency costs in the flanker task. Finally, more novel cues reduced the shift towards excitation in OA, partly restoring their E:I balance, and diminishing congruency costs. These findings demonstrate that the broadband shape of the EEG spectrum varies dynamically in a manner that is predictive of subsequent behavior. They also expand our understanding of how neural communication shapes cognition in YA and OA and have implications for neuroscientific models of cognitive processing and age-related cognitive decline.
Acid enhances salt taste by activating the epithelial sodium channel
Authors: Collier Daniel M. , Peterson Zerubbabel J. , Ryan Alan J. , Williford Noah N. , Benson Christopher J. , Snyder Peter M.
Published: April 2023

Salt is often used to enhance the flavor of foods and drinks, and in turn, some foods and drinks may intensify the taste of salt. For example, highly acidic carbonated beverages sometimes accompany salty snacks, and margaritas made with acidic citrus are served in salt-rimmed glasses. However, whether and how acid might enhance salt taste remain unknown. Epithelial sodium channels (ENaC) in tongue taste cells detect dietary sodium. We found that acid irreversibly increased ENaC channel activity, with half-maximal activation occurring at pH 2.6. Acid altered ENaC gating by increasing the rate of channel opening and reducing the rate of channel closing. Acidic beverages Coca-Cola® and Pepsi® (pH 2.2-2.4) also stimulated ENaC current but Diet Coke® (pH 3.2) did not. In humans, we found that acid reduced the sodium taste detection threshold. These findings identify a functional interplay between dietary sodium and acid--by modulating ENaC gating, acid enhances salt taste.
Charting spatial ligand-target activity using Renoir
Authors: Rao Narein , Kumar Tanush , Pai Rhea , Mishra Archita , Ginhoux Florent , Chan Jerry , Sharma Ankur , Zafar Hamim
Published: April 2023

The advancement of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics has enabled the inference of cellular interactions in a tissue microenvironment. Despite the development of cell-cell interaction inference methods, there is a lack of methods capable of mapping the influence of ligands on downstream target genes across a spatial topology with specific cell type composition, with the potential to shed light on niche-specific relationship between ligands and their downstream targets. Here we present Renoir for charting the ligand-target activities across a spatial topology and delineating spatial communication niches harboring specific ligand-target activities. Renoir also spatially maps pathway-level activity of ligand-target genesets and identifies domain-specific ligand-target activities. Across spatial datasets with varying resolution (spot to single-cell) ranging from development to disease, Renoir inferred cellular niches with distinct ligand-target interactions, spatially mapped pathway activities, and identified context-specific novel cell-cell interactions. Renoir uncovers biological insights and therapeutically-relevant cellular crosstalk from spatial transcriptomics data.
The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation
Authors: Wheeler Benjamin D , Gagnon John D , Zhu Wandi S , Muñoz-Sandoval Priscila , Wong Simon K , Simeonov Dimitre S , Li Zhongmei , Debarge Rachel , Spitzer Matthew , Marson Alexander , Ansel K Mark
Published: April 2023

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong andListeria monocytogenesinfection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specificin vivofunction to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.
Label-free Quantification of Host-Cell Protein Impurity in a Recombinant Hemoglobin Reference Material
Authors: Henrion André , Arsene Cristian , Liebl Maik , O’Connor Gavin
Published: April 2023

Quantitative analysis depends on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host-cell proteins (HCPs) in bioengineered proteins. For example, hemoglobin-A2 (HbA2) is being used as obtained through overexpression inE.coli.Two different materials had been produced: natural, and U-15N-labeled HbA2. For quantification of impurity, precursorion (MSl-) intensities were integrated over allE.coli-proteins identified, and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results forE.coli-cell lysate, which had been spiked at known mass-ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results could also be obtained when the complexity of the calibrator is reduced to a mix of nine proteins, and a minimum of five proteins is estimated to be sufficient to keep the sampling error below l5%. For the studied materials, HbA2-mass fractions of 916±15 mg/g and 922±11 mg/g were found. Value assignment by LFQ thus contributes 1-2% to the overall uncertainty of HbA2-quantification when these materials are used as calibrators. Further purification of the natural HbA2yielded 999.1± 0.15 mg/g, corresponding to ≈ 0.2% of uncertainty contribution, though at a significant loss of material. If an overall-uncertainty of 5% is acceptable for protein-quantification, working with the original materials would definitely be viable, therefore.
Lepidoptera genomics based on 88 chromosomal reference sequences informs population genetic parameters for conservation
Authors: Bortoluzzi Chiara , Wright Charlotte J. , Lee Sangjin , Cousins Trevor , Genez Thiago A. L. , Thybert David , Martin Fergal J. , Haggerty Leanne ,   The Darwin Tree of Life Project Consortium, Blaxter Mark , Durbin Richard
Published: April 2023

Butterflies and moths (Lepidoptera) are one of the most ecologically diverse and speciose insect orders, with more than 157,000 described species. However, the abundance and diversity of Lepidoptera are declining worldwide at an alarming rate. As few Lepidoptera are explicitly recognised as at risk globally, the need for conservation is neither mandated nor well-evidenced. Large-scale biodiversity genomics projects that take advantage of the latest developments in long-read sequencing technologies offer a valuable source of information. We here present a comprehensive, reference-free, whole-genome, multiple sequence alignment of 88 species of Lepidoptera. We show that the accuracy and quality of the alignment is influenced by the contiguity of the reference genomes analysed. We explored genomic signatures that might indicate conservation concern in these species. In our dataset, which is largely from Britain, many species, in particular moths, display low heterozygosity and a high level of inbreeding, reflected in medium (0.1 - 1 Mb) and long (> 1 Mb) runs of homozygosity. Many species with low inbreeding display a higher masked load, estimated from the sum of rejected substitution scores at heterozygous sites. Our study shows that the analysis of a single diploid genome in a comparative phylogenetic context can provide relevant genetic information to prioritise species for future conservation investigation, particularly for those with an unknown conservation status.
Signal-amplification for cell-free biosensors, an analog-to-digital converter
Authors: Lopes Franco Rafael Augusto , Brenner Gabriel , Bertolazzi Zocca Vitória Fernanda , de Paiva Gabriela Barbosa , Lima Rayane Nunes , Rech Elibio Leopoldo , Costa Ribeiro Lins Milca Rachel da , Pedrolli Danielle Biscaro
Published: April 2023

Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and Sars-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficientl contained leakage, boosted the expression fold-change from OFF to ON, and decreased the detection limit of the switches by three to four orders of magnitude. Ultimately, the integrase circuit converted the analog switches’ signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.

GRAPHICAL ABSTRACT
Canalisation and plasticity on the developmental manifold ofCaenorhabditis elegans
Authors: Jordan David J , Miska Eric A
Published: April 2023

How do the same mechanisms that faithfully regenerate complex developmental programs in spite of environmental and genetic perturbations also permit responsiveness to environmental signals, adaptation, and genetic evolution? Using the nematodeCaenorhabditis elegansas a model, we explore the phenotypic space of growth and development in various genetic and environmental contexts. Our data are growth curves and developmental parameters obtained by automated microscopy. Using these, we show that among the traits that make up the developmental space, correlations within a particular context are predictive of correlations among different contexts. Further we find that the developmental variability of this animal can be captured on a relatively low dimensionalphenoptypic manifoldand that on this manifold, genetic and environmental contributions to plasticity can be deconvolved independently. Our perspective offers a new way of understanding the relationship between robustness and flexibility in complex systems, suggesting that projection and concentration of dimension can naturally align these forces as complementary rather than competing.
Gut microbiome-based prediction of autoimmune neuroinflammation
Authors: Steimle Alex , Neumann Mareike , Grant Erica T. , Willieme Stéphanie , De Sciscio Alessandro , Parrish Amy , Ollert Markus , Miyauchi Eiji , Soga Tomoyoshi , Fukuda Shinji , Ohno Hiroshi , Desai Mahesh S.
Published: April 2023

Gut commensals are linked to neurodegenerative diseases, yet little is known about causal and functional roles of microbial risk factors in the gut–brain axis. Here, we employed a pre-clinical model of multiple sclerosis in mice harboring distinct complex microbiotas and six defined strain combinations of a functionally-characterized synthetic human microbiota. Discrete microbiota compositions resulted in different probabilities for development of severe autoimmune neuroinflammation. Nevertheless, assessing presence or the relative abundances of a suspected microbial risk factor failed to predict disease courses across different microbiota compositions. Importantly, we found considerable inter-individual disease course variations between mice harboring the same microbiota. Evaluation of multiple microbiome-associated functional characteristics and host immune responses demonstrated that the immunoglobulin A-coating index ofBacteroides ovatusbefore disease onset is a robust individual predictor for disease development. Our study highlights that the “microbial risk factor” concept needs to be seen in the context of a given microbial community network, and host-specific responses to that community must be considered when aiming for predicting disease risk based on microbiota characteristics.
Defining the mechanisms of action and resistance to the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain models
Authors: Phadke Manali S. , Li Jiannong , Chen Zhihua , Rodriguez Paulo C. , Mandula J.K. , Karapetyan Lilit , Forsyth Peter A. , Chen Y. Ann , Smalley Keiran S.M.
Published: April 2023

Background

Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma it remains unclear whether their mechanisms of action and resistance overlap.

Methods

We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion and ELISPOT assays were used to demonstrate the unique role of CD4+ T cell help in the anti-tumor effects of the anti-PD-1+LAG-3 combination. Tetramer assays confirmed the loss of CD8+ tumor-reactive T cells in brain tumors resistant to the anti-PD-1+LAG-3 combination.

Results

The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+ regulatory CD4+ cells (Tregs), fewer activated CD4+ T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+ T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+ T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+ T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+ T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+ T cell depletion was associated with fewer activated (CD69+) CD8+ T cells, impaired IFNγ release and increased numbers of myeloid-derived suppressor cells (MDSCs) but, conversely, increased numbers of activated CD8+ T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors. Analysis of relapsing melanoma brain metastases from anti-PD-1+LAG-3 treated mice showed an increased accumulation of MDSCs and a loss of gp100+ tumor reactive CD8+ T cells. An analysis of the inferred cell-cell interactions from the scRNA-seq data suggested the MDSCs interacted with multiple subsets of T cells in a bi-directional manner.

Conclusions

Together these studies suggest that these two clinically relevant ICI combinations have differential effects upon CD4+ T cell polarization, which in turn, impacted cytotoxic CD8+ T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.