bluuebunny/crossref_metadata_2025 huggingface dataset:
Pangenome-based dynamic trajectories of intracellular gene transfers in Poaceae unveil a high rate of unceasing integration and selective retention in Triticeae
Authors: Chen Yongming , Guo Yiwen , Xie Xiaoming , Wang Zihao , Miao Lingfeng , Yang Zhengzhao , Jiao Yuannian , Xie Chaojie , Liu Jie , Hu Zhaorong , Xin Mingming , Yao Yingyin , Ni Zhongfu , Sun Qixin , Peng Huiru , Guo Weilong
Published: October 2022

Intracellular gene transfers (IGTs) between the nucleus and organelles, including plastids and mitochondria, constantly reshapes the nuclear genome during evolution. Despite the substantial contribution of IGTs to genome variation, the dynamic trajectories of IGTs at the pangenomic level remain elusive. Here, we propose a novel approach, IGTminer, to map the evolutionary trajectories of IGTs by collinearity and gene reannotation across multiple genome assemblies. IGTminer was applied to create a nuclear organelle gene (NOG) map across 67 genomes covering 15 Poaceae species, including important crops, revealing the polymorphisms and trajectory dynamics of NOGs. The NOGs produced were verified by experimental evidence and resequencing datasets. We found that most of the NOGs were recently transferred and lineage specific, and that Triticeae species tended to have more NOGs than other Poaceae species. Wheat had a higher retention rate of NOGs than maize and rice, and the retained NOGs were likely involved in the photosynthesis and translation pathways. Large numbers of NOG clusters were aggregated in hexaploid wheat during two rounds of polyploidization and contributed to the genetic diversities among modern wheat varieties. Finally, we proposed a radiocarbon-like model illustrating the transfer and elimination dynamics of NOGs, highlighting the unceasing integration and selective retention of NOGs over evolutionary time. In addition, we implemented an interactive webserver for NOG exploration in Poaceae. In summary, this study provides new resources and clues for the roles of IGTs in shaping inter- and intraspecies genome variation and driving plant genome evolution.
Decoding brain basis of laughter and crying in natural scenes
Authors: Nummenmaa Lauri , Malèn Tuulia , Nazari-Farsani Sanaz , Seppälä Kerttu , Sun Lihua , Karlsson Henry K. , Hudson Matthew , Hirvonen Jussi , Sams Mikko , Scott Sophie , Putkinen Vesa
Published: October 2022

Laughter and crying are universal signals of prosociality and distress, respectively. Here we investigated the functional brain basis of perceiving laughter and crying using naturalistic functional magnetic resonance imaging (fMRI) approach. We measured haemodynamic brain activity evoked by laughter and crying in three experiments with 100 subjects in each. The subjects i) viewed a 20-minute medley of short video clips, and ii) 30 minutes of a full-length feature film, and iii) listened to 15 minutes of a radio play that all contained bursts of laughter and crying. Intensity of laughing and crying in the videos and radio play was annotated by independent observes, and the resulting time series were used to predict hemodynamic activity to laughter and crying episodes. Multivariate pattern analysis (MVPA) was used to test for regional selectivity in laughter and crying evoked activations. Laughter induced widespread activity in ventral visual cortex and superior and middle temporal and motor cortices. Crying activated thalamus, cingulate cortex along the anterior-posterior axis, insula and orbitofrontal cortex. Both laughter and crying could be decoded accurately (66-77% depending on the experiment) from the BOLD signal, and the voxels contributing most significantly to classification were in superior temporal cortex. These results suggest that perceiving laughter and crying engage distinct neural networks, whose activity suppresses each other to manage appropriate behavioral responses to others’ bonding and distress signals.

Significance statement

Laughter and crying are universal signals of prosociality and distress, respectively. They occur in complex, dynamic social settings with variable and dynamically evolving time courses. Here we used functional magnetic resonance imaging experiments and statistical pattern recognition for disentangling the neural systems that encode laughter and crying signals from dynamic and highly naturalistic scenes. These results show that separable neural circuits are engaged in processing distinct types of social attachment cues, and that pattern recognition during dynamic scene perception allows reliable separation of laughter and crying evoked activation patterns. Coordinated activity of these networks allows managing appropriate behavioral responses to others’ bonding and distress signals.
Investigating the oral microbiome in retrospective and prospective cases of prostate, colon, and breast cancer
Authors: Nearing Jacob T. , DeClercq Vanessa , Langille Morgan G.I.
Published: October 2022

The human microbiome has been proposed as a useful biomarker for several different human diseases including various cancers. To answer this question, we examined salivary samples from two Canadian population cohorts, the Atlantic Partnership for Tomorrow’s Health project (PATH) and Alberta’s Tomorrow Project (ATP). Sample selection was then divided into both a retrospective and prospective case control design examining individuals with prostate, breast, or colon cancer. In total 89 retrospective and 260 prospective cancer cases were matched to non-cancer controls and saliva samples were sequenced using 16S rRNA gene sequencing to compare bacterial diversity, and taxonomic composition. We found no significant differences in alpha or beta diversity across any of the three cancer types and two study designs. Although retrospective colon cancer samples did show evidence on visual clustering in weighted beta diversity metrics. Differential abundance analysis of individual taxon showed several taxa that were associated with previous cancer diagnosis in all three groupings within the retrospective study design. However, only one genus (Ruminococcaceae UCG-014) in breast cancer and one ASV (Fusobacterium periodonticum) in colon cancer was identified by more than one differential abundance (DA) tool. In prospective cases of disease three ASVs were associated with colon cancer, one ASV with breast cancer, and one ASV with prostate cancer. None overlapped between the two different study cohorts. Attempting to identify microbial signals using Random Forest classification showed relatively low levels of signal in both prospective and retrospective cases of breast and prostate cancer (AUC range: 0.394-0.665). Contrastingly, colon cancer did show signal in our retrospective analysis (AUC: 0.745) and in one of two prospective cohorts (AUC: 0.717). Overall, our results indicate that it is unlikely that reliable oral microbial biomarkers of disease exist in the context of both breast and prostate cancer. However, they do suggest that further research into the relationship between the oral microbiome and colon cancer could be fruitful. Particularly in the context of early disease progression and risk of cancer development.
Development of SARS-CoV-2 replicons for the ancestral virus and variant of concern Delta for antiviral screening
Authors: Erdmann Maximilian , Williamson Maia Kavanagh , Jearanaiwitayakul Tuksin , Bazire James , Matthews David A. , Davidson Andrew D.
Published: October 2022

SARS-CoV-2 is the aetiologic agent of COVID-19 and the associated ongoing pandemic. As the pandemic has progressed, Variants of Concern (VOC) have emerged with lineage defining mutations. Using a SARS-CoV-2 reverse genetic system, based on transformation associated recombination in yeast, a series of replicons were produced for the ancestral Wuhan virus and the SARS-CoV-2 VOC Delta in which different combinations of the Spike, membrane, ORF6 and ORF7a coding sequences were replaced with sequences encoding the selectable marker puromycin N-acetyl transferase and reporter proteins (Renillaluciferase, mNeonGreen and mScarlet). Replicon RNAs were replication competent in African green monkey kidney (Vero E6) derived cells and a range of human cell lines, with a Vero E6 cell line expressing ACE2 and TMPRSS2 showing much higher transfection efficiency and overall levels ofRenillaluciferase activity. The replicons could be used for transient gene expression studies, but cell populations that stably maintained the replicons could not be propagated. Replication of the transiently expressed replicon RNA genomes was sensitive to remedesivir, providing a system to dissect the mechanism of action of antiviral compounds.
Maternal body condition and season influence RNA deposition in the oocytes of alfalfa leafcutting bees (Megachile rotundata)
Authors: Hagadorn Mallory A. , Hunter Frances K. , DeLory Tim , Johnson Makenna M. , Pitts-Singer Theresa L. , Kapheim Karen M.
Published: October 2022

Maternal effects are an important source of phenotypic variance, whereby females influence offspring developmental trajectory beyond direct genetic contributions, often in response to changing environmental conditions. However, relatively little is known about the mechanisms by which maternal experience is translated into molecular signals that shape offspring development. One such signal may be maternal RNA transcripts (mRNAs and miRNAs) deposited into maturing oocytes. These regulate the earliest stages of development of all animals, but are understudied in most insects. Here we investigated the effects of female internal (body condition) and external (time of season) environmental conditions on maternal RNA in the maturing oocytes and 24 hr old eggs of alfalfa leafcutting bees. Using gene expression and WGCNA analysis, we found that females adjust the quantity of mRNAs related to protein phosphorylation, transcriptional regulation, and nuclease activity deposited into maturing oocytes in response to both poor body condition and shorter day lengths that accompany the late season. However, the magnitude of these changes was higher for time of season. Females also adjusted miRNA deposition in response to seasonal changes, but not body condition. We did not observe significant changes in maternal RNAs in response to either body condition or time of season in 24-hr-old eggs, which were past the maternal-to-zygotic transition. Our results suggest that females adjust the RNA transcripts they provide for offspring to regulate development in response to both internal and external environmental cues. Variation in maternal RNAs may, therefore, be important for regulating offspring phenotype in response to environmental change.
The unifying catalytic mechanism of the RING-between-RING E3 ligase family
Authors: Wang Xiangyi S. , Cotton Thomas R. , Trevelyan Sarah J. , Richardson Lachlan W. , Lee Wei Ting , Silke John , Lechtenberg Bernhard C.
Published: October 2022

The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, in combination with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric mechanism by distinct ubiquitin linkages that suggests that RBRs employ a feed-forward mechanism accelerating their signalling pathways. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 bivalent zinc cluster that may represent a specific adaptation enabling HOIL-1’s atypical substrate specificity.
Polyphasic circadian neural circuits drive differential activities in multiple downstream rhythmic centers
Authors: Liang Xitong , Holy Timothy E. , Taghert Paul H.
Published: October 2022

Circadian clocks align various behaviors such as locomotor activity, sleep/wake, feeding, and mating to times of day that are most adaptive. How rhythmic information in pacemaker circuits is translated to neuronal outputs is not well understood. Here we used brain-wide, 24-hrin vivocalcium imaging in theDrosophilabrain and searched for circadian rhythmic activity among identified clusters of dopaminergic (DA) and peptidergic neuroendocrine (PNE) neurons. Such rhythms were widespread and imposed by the PERIOD-dependent clock activity within the ∼150 cell circadian pacemaker network. The rhythms displayed either a Morning, an Evening, or Mid-Day phase. Different sub-groups of circadian pacemakers imposed neural activity rhythms onto different downstream non-clock neurons. Outputs from the canonical M and E pacemakers converged to regulate DA-PPM3 and DA-PAL neurons. E pacemakers regulate the Evening-active DA-PPL1 neurons. In addition to these canonical M and E oscillators, we present evidence for a third dedicated phase occurring at Mid-Day (MD): the l-LNv pacemakers present the MD activity peak and they regulate the MD-active DA-PPM1/2 neurons and three distinct PNE cell types. Thus, theDrosophilacircadian pacemaker network is a polyphasic rhythm generator. It presents dedicated M, E and MD phases that are functionally transduced as neuronal outputs to organize diverse daily activity patterns in downstream circuits.
Renegade Bacterial Genetic Sequences in a Stealth Adapted Virus: Biological and Diagnostic Implications
Authors: Martin W. John
Published: October 2022

There are major differences between viruses, bacteria, and eukaryotic cells in the structuring of their genomes, modes of replication, and capacity to horizontally transfer genetic sequences. DNA sequencing studies on a virus cultured from a patient with chronic fatigue syndrome (CFS) have confirmed a previously underappreciated capacity of certain viruses to capture and transfer bacterial and cellular genetic sequences between eukaryotic cells as part of the infectious process. The virus originated from an African green monkey simian cytomegalovirus (SCMV). It is termed a stealth adapted virus since infection is not accompanied by inflammation. The immune evasion is attributed to the loss and mutation of the genes coding for the relatively few components that are normally targeted by the cellular immune system. This article provides further elucidation of the origins of many of the bacterial-derived genetic sequences present in the virus. There are multiple clones with close but non-identical sequence alignments with different genomic regions of theOchrobactrum quorumnocensA44 species of bacteria. Another set of clones matched most closely to diverse genomic regions ofMycoplasma fermentansbacteria. The sequences of several other clones could only be approximately aligned to those of different types of bacteria. The sequence of clone 3B513 is consistent with genetic contributions from the genomes of several types of bacteria. The term viteria refers to viruses with bacteria-derived genetic sequences. They are the likely primary cause of CFS and autism, and to act as major cofactors in many illnesses, including AIDS. As a more general phenomenon viteria with different types of renegade bacterial sequences can lead to the mistaken diagnoses of bacterial rather than viral diseases. It is important to genetically sequence additional stealth adapted viruses from patients with a wide range of illnesses, including those currently being attributed to Mycoplasma, Borrelia, or Streptococcal infections.
Human papillomavirus intermittence, redetections, and associated risk of cytological abnormalities in the Ludwig-McGill cohort study of adult women
Authors: Malagón Talía , Trottier Helen , El-Zein Mariam , Villa Luisa L , Franco Eduardo ,   the Ludwig-McGill Cohort Study
Published: October 2022

Introduction

We assessed the incidence of redetection with the same human papillomavirus (HPV) genotype, predictors of first HPV detections and redetections, and prevalence of cytological lesions during HPV redetections.

Methods

The Ludwig-McGill cohort study followed women aged 18-60 years from São Paulo, Brazil in 1993-1997 for up to 10 years. Women provided cervical samples for cytology testing and HPV DNA testing at each visit. A redetection was defined as a recurring genotype-specific HPV positive result after one or more intervening negative visits. Predictors of genotype-specific redetection were assessed using adjusted hazard ratios (aHR) with Cox regression modeling.

Results

2184 women contributed 2368 incident HPV genotype-specific first detections and 308 genotype-specific redetections over a median follow-up of 6.5 years. The cumulative incidence of redetection with the same genotype was 7% at 1 year and 15% at 5 years after the loss of positivity of the first detection. Neither age (aHR 0.90, 95%CI 0.54-1.47 for ≥45y vs. <25y) nor new sexual partner acquisition (aHR 0.98, 95%CI 0.70-1.35) were statistically associated with genotype-specific redetection. High-grade squamous intraepithelial lesion prevalence was similar during first HPV detections (2.9%) and redetection (3.2%).

Conclusions

Our findings suggest many HPV redetections were likely reactivations of latent recurring infections.
Benchtop evaluation of a double stent retriever thrombectomy technique for acute ischemic stroke treatment
Authors: Hofmeister Jeremy , Bernava Gianmarco , Rosi Andrea , Reymond Philippe , Brina Olivier , Muster Michel , Lovblad Karl-Olof , Machi Paolo
Published: October 2022

Background

A mechanical thrombectomy technique using a double stent retriever (DSR) approach has been reported for the treatment of patients with acute ischemic stroke. The purpose of this study was to perform a benchtop evaluation of the mechanism of action and efficacy of a DSR approach compared to a single stent retriever approach.

Methods

In vitromechanical thrombectomy procedures were performed in a vascular phantom reproducing a M1-M2 occlusion with two different clot analog consistencies (soft and hard). We compared the DSR approach to the single stent retriever approach and recorded the recanalisation rate, distal embolization, and retrieval forces of each mechanical thrombectomy procedure.

Results

The DSR approach achieved a higher recanalization rate and lower embolic complications compared to the single stent retriever approach. This seems to stem from two facts: the greater probability of targeting the correct artery with two stents in the case of bifurcation occlusion, and an improved clot capture mechanism using the DSR approach. However, the DSR was associated with an increased initial retrieval force.

Conclusion

In vitroevaluation of the mechanism of action of the DSR provided explanations that appear to support the high efficacy of such an approach in patient cohorts and could help operators when selecting the optimal mechanical thrombectomy strategy in cases of arterial occlusions difficult to treat with a single stent retriever.
Global neuropathologic severity of Alzheimer’s disease and locus coeruleus vulnerability influences plasma phosphorylated tau levels
Authors: Murray Melissa E. , Moloney Christina M. , Kouri Naomi , Syrjanen Jeremy A. , Matchett Billie J. , Rothberg Darren M. , Tranovich Jessica F. , Hicks Sirmans Tiffany N. , Wiste Heather J. , Boon Baayla D. C. , Nguyen Aivi T. , Reichard R. Ross , Dickson Dennis W. , Lowe Val J. , Dage Jeffrey L , Petersen Ronald C. , Jack Clifford R. , Knopman David S. , Vemuri Prashanthi , Graff-Radford Jonathan , Mielke Michelle M.
Published: October 2022

Advances in ultrasensitive detection of phosphorylated tau (p-tau) in plasma has enabled the use of blood tests to measure Alzheimer’s disease (AD) biomarker changes. Examination of postmortem brains of participants with antemortem plasma p-tau levels remains critical to understanding comorbid and AD-specific contribution to these biomarker changes. We analyzed 35 population-based Mayo Clinic Study of Aging participants with plasma p-tau at threonine181 and threonine217 (p-tau181, p-tau217) available within 3 years of death. Autopsied participants included cognitively unimpaired, mild cognitive impairment, AD dementia, and non-AD neurodegenerative disorders. Global neuropathologic scales of tau, amyloid-β, TDP-43, and cerebrovascular disease were examined. Regional digital pathology measures of tau (phosphorylated threonine181 and 217 [pT181, pT217]) and amyloid-β (6F/3D) were quantified in hippocampus and parietal cortex. Neurotransmitter hubs reported to influence development of tangles (nucleus basalis of Meynert) and amyloid-β plaques (locus coeruleus) were evaluated. The strongest regional associations were with parietal cortex for tau burden (p-tau181 R=0.55, p=0.003; p-tau217 R=0.66, p<0.001) and amyloid-β burden (p-tau181 R=0.59, p<0.001; p-tau217 R=0.71, p<0.001). Linear regression analysis of global neuropathologic scales explained 31% of variability in plasma p-tau181 (R2=0.31) and 59% in plasma p-tau217 (R2=0.59). Neither TDP-43 nor cerebrovascular disease global scales independently contributed to variability. Global scales of tau pathology (β-coefficient=0.060, p=0.016) and amyloid-β pathology (β-coefficient=0.080, p<0.001) independently predicted plasma p-tau217 when modeled together with co-pathologies, but only amyloid-β (β-coefficient=0.33, p=0.021) significantly predicted plasma p-tau181. While nucleus basalis of Meynert neuron count/mm2was not associated with plasma p-tau levels, a lower locus coeruleus neuron count/mm2was associated with higher plasma p-tau181 (R=-0.50, p=0.007) and higher plasma p-tau217 (R=-0.55, p=0.002). Cognitive scores (R2=0.31-0.34) were predicted by the global tau scale, but not by the global amyloid-β scale or plasma p-tau when modeled simultaneously. Higher soluble plasma p-tau levels may be the result of an intersection between insoluble deposits of amyloid-β and tau accumulation in brain and may be associated with locus coeruleus degeneration.
Risk factors for suicide following treatment with electroconvulsive therapy: A nationwide study of 11,780 patients
Authors: Spanggård Anders , Rohde Christopher , Østergaard Søren Dinesen
Published: October 2022

Objectives

Despite the well-established anti-suicidal effect of electroconvulsive therapy (ECT), patients receiving ECT remain at high risk of dying from suicide. In the present study, we aimed to quantify this risk and identify risk factors for suicide among patients receiving ECT.

Methods

We used nationwide Danish registers to identify all patients that initiated ECT between 2006 and 2016. These patients were matched on sex and age to 10 reference individuals from the general Danish population. First, we compared 2-year suicide risk between patients initiating ECT and the matched reference individuals. Second, we investigated if any patient characteristics were associated with suicide following ECT via Cox proportional-hazards regression.

Results

A total of 11,780 patients receiving ECT and 117,800 reference individuals were included in the analyses. Among the patients receiving ECT, 161 (1.4%) died from suicide within two years. Compared to the reference individuals, patients receiving ECT had a substantially elevated suicide rate (Hazard rate ratio (HRR)=44.5, 95%CI=31.1-63.6). Among those receiving ECT, we identified the following risk factors for suicide: Male sex (HRR=2.3, 95%CI=1.7-3.1), age 60-70 years (HRR=1.6, 95%CI=1.0-2.6), Medium-term higher education (HRR=1.5, 95%CI=1.0-2.2); Long-term higher education (HRR=1.9, 95%CI=1.1-3.1), history of substance use disorder (HRR=2.0, 95%CI=1.4-2.8) and history of intentional self-harm/suicide attempt (HRR=4.0, 95%CI=2.8-5.8).

Conclusion

Among patients receiving ECT, those who are male, aged 60-70 years, have mediumterm to long-term higher education, or have a history of substance use disorder or intentional self-harm/suicide attempt, are at particularly elevated risk of suicide. These findings may guide initiatives to reduce the risk of suicide.
WITHDRAWN: Post-acute symptoms four months after SARS-CoV-2 infection during the Omicron period: a nationwide Danish questionnaire study
Authors: Spiliopoulos Lampros , Sørensen Anna Irene Vedel , Bager Peter , Nielsen Nete Munk , Hansen Jørgen Vinsløv , Koch Anders , Meder Inger Kristine , Videbech Poul , Ethelberg Steen , Hviid Anders
Published: October 2022

Withdrawal Statement

This manuscript has been removed because it included data obtained through a copyrighted questionnaire. The survey described in the manuscript was launched in haste during the pandemic and the authors were not aware of the copyright protection and acted in good faith. Attempts to obtain permission to use the questionnaire retrospectively were rejected by the copyright holders. The first author was not involved in the decision to include the copyrighted questionnaire, in any other aspects of the design of the survey or in the data collection and therefore bears no responsibility for this mishap. The senior author Anders Hviid takes full responsibility.
CRISPR-Cas phage defense systems and prophages inCandidatusAccumulibacter
Authors: Deng Xuhan , Yuan Jing , Chen Liping , Chen Hang , Wei Chaohai , Nielsen Per H. , Wuertz Stefan , Qiu Guanglei
Published: October 2022

CandidatusAccumulibacter is a key genus of polyphosphate-accumulating organisms (PAOs) found in laboratory- and full-scale wastewater treatment systems, mediating enhanced biological phosphorus removal (EBPR). However, little is known about their ability to resist phage infection. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in diverseCa.Accumulibacter taxa (43 in total, including 10 newly recovered genomes). Fourty complete CRISPR loci were identified in 28 genomes, falling into 6 subtypes. The occurrence and distribution of CRISPR-Cas systems did not follow a vertical evolutionary relationship. Phylogenetic analyses of thecasgenes and direct repeats (DRs) suggested that the CRISPR-Cas systems were likely acquired via horizontal gene transfer, with acquisition rates higher than those of speciation, rendering differentCa.Accumulibacter distinct adaptivity to phage predations. 2448 spacers were identified, 67 of them matched to known phages. Major differences were observed among the numbers of spacers for differentCa.Accumulibacter, showing unique phages that could be resisted by different members. A comparison of the spacers in genomes having the samecasgene but from distinct geographical locations indicated that habitat isolation may have resulted in the acquisition of different spacers by differentCa. Accumulibacter. Metagenomic analysis allowed the identification of 26 viral contigs (18 are Caudovirales members) in 6 metagenomic datasets from three lab-scale enrichment reactors, matching to 73 spacers in 10Ca.Accumulibacter genomes in these reactors, showing the specific immunity of theseCa.Accumulibacter. Metatranscriptomic analyses showed the activity of the CRISPR-Cas system under both anaerobic and aerobic conditions. Extra efforts were made to identify prophages inCa.Accumulibacter genomes. In total, 133 prophage regions were identified. Twenty-seven of them were classified as potentially active prophages. Three prophages (all are Caudovirales members, in DS2011, SCELSE-7IIH and SCELSE-5IIH, respectively) are readily activable. Differences in the occurrence of CRISPR-Cas systems and prophages inCa.Accumulibacter will lead to their distinct responses under phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages with combined experimental and bioinformatic methods in theCa.Accumulibacter lineage, providing new perspectives on the potential impacts of phages onCa.Accumulibacter and EBPR systems.
Permeation Across the Mycomembrane in Live Mycobacteria
Authors: Liu Zichen , Lepori Irene , Dalesandro Brianna E. , Chordia Mahendra D. , Guo Taijie , Sharpless Karl Barry , Dong Jiajia , Siegrist M. Sloan , Pires Marcos M.
Published: October 2022

The general lack of permeability of small molecules observed forMycobacterium tuberculosis(Mtb) is most commonly ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. Nonetheless, there is limited information on the types of molecular scaffolds that can readily permeate past the mycomembrane of mycobacteria. To address this, we describe a novel assay that combines metabolic tagging of the peptidoglycan scaffold, which sits directly beneath the mycomembrane, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis inMycobacterium smegmatisandMtb. A small panel of molecules was tested and we found a large range in the permeability profile of molecules. Interestingly, the general trend is similar across the two types of mycobacteria, with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria. The methods described, which do not require genetic manipulation, can be generally adopted to other species for which envelope permeability is treatment-limiting.
IGFBP2 expressing midlobular hepatocytes preferentially contribute to liver homeostasis and regeneration
Authors: Lin Yu-Hsuan , Wei Yonglong , Wang Yunguan , Pagani Chase A. , Li Lin , Zhu Min , Wang Zixi , Hsieh Meng-Hsiung , Zhang Yu , Sharma Tripti , Wang Tao , Zhu Hao
Published: October 2022

While midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated aIgfbp2-CreERknockin strain, which specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21% to 41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with DDC, IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration.

Highlights

Lineage tracing showed that midlobular hepatocytes proliferate during homeostasis.

Zone 2 cells were protected from portal and centrilobular injuries and replaced lost hepatocytes.

Fasting induced significant changes in liver zonation.

eTOC statement

The liver consists of different zones with spatial heterogeneity in their metabolic functions. Here, a newIgfbp2-CreERline enabled direct tracing of midlobular hepatocytes, which showed that IGFBP2+ cells serve as a source of new hepatocytes during normal homeostasis and regeneration.
mTORC2 Interactome and Localization Determine Aggressiveness of High-Grade Glioma Cells through Association with Gelsolin
Authors: Chantaravisoot Naphat , Wongkongkathep Piriya , Kalpongnukul Nuttiya , Pacharakullanon Narawit , Kaewsapsak Pornchai , Ariyachet Chaiyaboot , Loo Joseph A. , Tamanoi Fuyuhiko , Pisitkun Trairak
Published: October 2022

The mTOR complex 2 (mTORC2) has been implicated as a key regulator of glioblastoma cell migration. However, the roles of mTORC2 in the migrational control process have not been entirely elucidated. Here we elaborate that active mTORC2 is crucial for GBM cell motility. Inhibition of mTORC2 impaired cell movement and negatively affected microfilaments and microtubules’ functions. We also aimed to characterize important players involved in the regulation of cell migration and other mTORC2-mediated cellular processes in GBM cells through the proteomic and bioinformatic analyses. Therefore, we quantitatively characterized the alteration of mTORC2 interactome under selective conditions using affinity-purification mass spectrometry (AP-MS) in glioblastoma. We demonstrated that changes in cell migration ability specifically altered mTORC2-associated proteins. Gelsolin (GSN) was identified as one of the most dynamic proteins. The mTORC2-GSN linkage was mostly highlighted in high-grade glioma cells and shown to connect functional mTORC2 to multiple proteins responsible for directional cell movement in GBM. GSN also mediated membrane localization of mTORC2. Loss of GSN disconnected mTORC2 from numerous cytoskeletal proteins but did not affect mTORC2 integrity. In addition, we reported 86 stable mTORC2 interactome involved with diverse molecular functions, predominantly cytoskeletal remodeling in GBM. Our findings help expand future opportunities for predicting the highly migratory phenotype of brain cancers in the clinical investigation.
Structural analysis of human papillomavirus E6 interactions with Scribble PDZ domains
Authors: Stewart Bryce Z. , Caria Sofia , Humbert Patrick O. , Kvansakul Marc
Published: October 2022

The cell polarity regulator Scribble has been shown to be a critical regulator of the establishment and development of tissue architecture, and its dysregulation promotes or suppresses tumor development in a context dependent manner. Scribble activity is subverted by numerous viruses. This includes human papillomaviruses (HPVs), who target Scribble via the E6 protein. Binding of E6 from high-risk HPV strains to Scribble via a C-terminal PDZ binding motif leads to Scribble degradationin vivo. However, the precise molecular basis for Scribble-E6 interactions remains to be defined. We now show that Scribble PDZ1 and PDZ3 are the major interactors of HPV E6 from multiple high-risk strains, with each E6 protein displaying a unique interaction profile. We then determined crystal structures of Scribble PDZ1 and PDZ3 domains in complex with the PBM motifs of E6 from HPV strains 16, 18 and 66. Our findings reveal distinct interaction patterns for each E6 PBM motif from a given HPV strain, suggesting that a complex molecular interplay exists that underpins the overt Scribble-HPV E6 interaction and controls E6 carcinogenic potential.
Spatial expression of an mRNA encoding Tie2-agonist in the capillary endothelium of the lung prevents pulmonary vascular leakage
Authors: Radloff Katrin , Gutbier Birgitt , Dunne Charlotte Maeve , Moradian Hanieh , Schwestka Marko , Gossen Manfred , Ahrens Katharina , Kneller Laura , Wang Yadong , Moga Akanksha , Gkionis Leonidas , Keil Oliver , Fehring Volker , Tondera Daniel , Giese Klaus , Santel Ansgar , Kaufmann Jörg , Witzenrath Martin
Published: October 2022

Angiopoietin ligands Ang1 and Ang2 and the Tie2 receptor tyrosine kinases form an endothelial signaling pathway regulating vascular homeostasis and controlling vessel permeability, inflammation and angiogenic responses. Whereas Ang1-mediated Tie2 activation reduces inflammation and endothelial permeability, its antagonist, Ang2 increases it. Increased plasma Ang2 levels are associated with poor outcomes in patients with acute lung injury (ALI), as well as in acute respiratory distress syndrome (ARDS).

In the study presented here we tested the effect of a novel synthetic, nucleoside-modified mRNA-76 encoding for a hyperactive Ang1 derived fusion protein (COMP-Ang1) on attenuating post-inflammation vascular leakage. COMP-Ang1 mRNA was formulated into a cationic lipid nanoparticle (cLNP) using an optimized mixture of three different lipids and a microfluidic mixing technology. After intravenous injection, the respective mRNA-loaded LNPs were found to be delivered predominantly to the endothelial cells of the lung, while sparing other vascular beds. Also, the specific multimeric folding of the COMP-Ang1 protein complex appeared to be pivotal for its activity in preventing vascular leakage and in restoring the alveolar-endothelial barrier function in the inflamed and injured pulmonary vasculature. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studiesin vitroandin vivoby systemic administration in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in an LPS-challenging model.
Endosomal Trafficking of Two Pore K+Efflux Channel TWIK2 to Plasmalemma Mediates NLRP3 Inflammasome Activation and Inflammatory Injury
Authors: Di Anke , Huang Long Shuang , Zhou Bisheng , Toth Peter T. , Krishnan Yamuna , Malik Asrar B.
Published: October 2022

Potassium efflux via the two pore K+channel TWIK2 is a requisite step for the activation of the NLRP3 inflammasome, however it is unclear how the efflux is activated in response to cues. Here we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in extrusion of K+ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP ligated purinergic receptor P2X7 prevented endosomal fusion with the plasmalemma and K+efflux and NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-deleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 abundance and activity on the cell surface and downstream activation of the NLRP3 inflammasome. Endosomal trafficking of TWIK2 to the plasmalemma is therefore a potential therapy target in acute or chronic inflammatory states.
Structures of the holo CRISPR RNA-guided transposon integration complex
Authors: Park Jung-Un , Tsai Amy Wei-Lun , Rizo Alexandrea N. , Truong Vinh H. , Wellner Tristan X. , Schargel Richard D. , Kellogg Elizabeth H.
Published: October 2022

CRISPR-associated transposons (CAST) are programmable mobile elements that insert large DNA cargo by an RNA-guided mechanism. Multiple conserved components act in concert at the target site through formation of an integration complex (transpososome). We reconstituted the type V-K CAST transpososome fromScytonema hofmannii(ShCAST) and determined the structure using cryo-EM. Transpososome architecture ensures orientation-specific association: AAA+ regulator TnsC has defined polarity and length, with dedicated interaction interfaces for other CAST components. Interestingly, transposase (TnsB)-TnsC interactions we observe contribute to stimulating TnsC’s ATP hydrolysis activity. TnsC deviates from previously observed helical configurations of TnsC, and target DNA does not track with TnsC protomers. Consequently, TnsC makes new, functionally important protein-DNA interactions throughout the transpososome. Finally, two distinct transpososome populations suggests that associations with the CRISPR effector are flexible. These ShCAST transpososome structures significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.
Synthetic lethal targeting ofTET2-mutant hematopoietic stem and progenitor cells by XPO1 inhibitors
Authors: Jing Chang-Bin , Prutsch Nicole , He Shuning , Zimmerman Mark W. , Landesman Yosef , Look A. Thomas
Published: October 2022

TET2inactivating mutations serve as initiating genetic lesions in the transformation of hematopoietic stem and progenitor cells (HSPCs). In this study, we analyzed known drugs in zebrafish embryos for their abilities to selectively killtet2-mutant HSPCsin vivo, and we found that the exportin 1 (XPO1) inhibitors, selinexor and eltanexor, selectively killtet2-mutant HSPCs. In serial replating colony assays, these small molecules were selectively active in killing murineTet2-deficient Lineage-, Sca1+, Kit+ (LSK) cells, and alsoTET2-inactivated human acute myeloid leukemia (AML) cells. Selective killing ofTET2-mutant HSPCs and human AML cells by these inhibitors was due to increased levels of apoptosis, without evidence of DNA damage based on increased γH2AX expression. The finding thatTET2loss renders HSPCs and AML cells selectively susceptible to cell death induced by XPO1 inhibitors provides preclinical evidence of selective activity of these drugs, justifying further clinical studies of these small molecules for the treatment ofTET2-mutant hematopoietic malignancies and to suppress clonal expansion in age-relatedTET2-mutant clonal hematopoiesis.
METTL7A and METTL7B are responsible forS-thiol methyl transferase activity in liver
Authors: Russell Drake A. , Chau Marvin K. , Shi Yuanyuan , Maldonato Benjamin J. , Totah Rheem A.
Published: October 2022

S-Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putativeS-adenosyl-L-methionine dependent membrane-associated phase II enzyme known as thiol methyltransferase (TMT). TMT has a broad substrate specificity and methylates the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMT’s role in theS-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase. METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to TMT. Yet, the historic TMT inhibitor, 2,3-dichloro-α-methylbenzylamine (DCMB), has no effect on the activity of METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, also acts as a thiolmethyltransferase. METTL7A exhibits similar biochemical properties to TMT, including inhibition by DCMB (IC50 1.2 µM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which we have renamed TMT1A1 and TMT1B1, respectively, that are responsible for TMT activity in liver microsomes.

Significance Statement

We identified METTL7A (TMT1A1) and METTL7B (TMT1B1) as the enzymes responsible for the microsomal alkyl thiol methyltransferase activity. These are the first two enzymes directly associated with microsomal TMT activity.S-Methylation of many commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible, will improve our understanding of the DMPK properties of alkyl- or phenolic-thiol-containing therapeutics.

Visual Abstract
Humanized CB1R and CB2R yeast biosensors enable facile screening of cannabinoid compounds
Authors: Mulvihill Colleen J. , Lutgens Josh , Gollihar Jimmy D. , Bachanová Petra , Marcotte Edward M. , Ellington Andrew D. , Gardner Elizabeth C.
Published: October 2022

Yeast expression of human G Protein Coupled Receptors (GPCRs) can be used as a biosensor platform for the detection of pharmaceuticals. The Cannabinoid receptors type 1 and 2 (CB1/2R) are of particular interest, given the cornucopia of natural and synthetic cannabinoids being explored as therapeutics. We show for the first time that engineering the N-terminus of CB1R allows for efficient signal transduction in yeast, and that engineering the sterol composition of the yeast membrane optimizes CB2R performance. Using the dual cannabinoid biosensors, large libraries of synthetic cannabinoids and terpenes could be quickly screened to elucidate known and novel structure-activity relationships, including compounds and trends that more selectively target each of the two receptors. The biosensor strains offer a ready platform for evaluating the activity of new synthetic cannabinoids, monitoring drugs of abuse, and developing molecules that target the therapeutically important CB2R receptor while minimizing psychoactive effects.
Extracellular vesicles from non-neuroendocrine SCLC cells promote adhesion and survival of neuroendocrine SCLC cells
Authors: Jimenez Lizandra , Stolzenbach Victor , Ozawa Patricia M. M. , Ramirez-Solano Marisol , Liu Qi , Sage Julien , Weaver Alissa M.
Published: October 2022

Small Cell Lung Cancer (SCLC) tumors are made up of distinct cell subpopulations, including neuroendocrine (NE) and non-NE cells. While secreted factors from non-NE SCLC cells have been shown to support the growth of the NE cells, the underlying molecular factors are not well understood. Here, we show that exosome-type small extracellular vesicles (SEVs) secreted from non-NE SCLC cells promote adhesion and survival of NE SCLC cells. Proteomic analysis of purified small EVs revealed that extracellular matrix (ECM) proteins and integrins are highly enriched in small EVs of non-NE cells whereas nucleic acid-binding proteins are enriched in small EVs purified from NE cells. Addition of select purified ECM proteins identified in purified EVs, specifically fibronectin, laminin 411, and laminin 511, were able to substitute for the role of non-NE-derived SEVs in promoting adhesion, survival, and tumorigenicity of NE SCLC cells. Those same proteins were differentially expressed by human SCLC subtypes. These data suggest that ECM-carrying SEVs secreted by non-NE cells play a key role in supporting SCLC tumor growth and survival.
Concurrent temporal patterning of neural stem cells in the fly visual system
Authors: Arain Urfa , Islam Ishrat Maliha , Valentino Priscilla , Erclik Ted
Published: October 2022

The temporal and spatial patterning of neural stem cells is a powerful mechanism by which to generate neural diversity in both vertebrate and invertebrate brains. In theDrosophilaoptic lobe, the neuroblasts (NBs) that generate the ∼120 neuronal cell types of the medulla are patterned by independent temporal and spatial inputs. In the temporal axis, a cascade of twelve transcription factors (TFs) are expressed in medulla NBs as they age. In the spatial axis, the neuroepithelium from which these NBs are generated is sub-divided into eight compartments by the expression of five additional TFs. Distinct neuronal types are generated by NBs based on their spatio-temporal address. Here, we describe a third major patterning axis that further diversifies neuronal fates in the medulla. We show that the symmetrically dividing neuroepithelial cells from which the medulla NBs are generated are temporally patterned by opposing gradients of the Imp and Syp RNA-binding proteins. Imp and Syp regulate the expression of a set of TFs in the neuroepithelium to confer NBs from the same spatio-temporal address with unique identities based on the developmental stage they are generated. We show that Imp and Syp differentially pattern NBs in the Vsx1-Hth spatio-temporal birth window to generate seven distinct neuronal cell types (Li2, TmY17, TmY15, Tm23, Pm3a, Pm3b and TmY12) in successive developmental windows. We further demonstrate that the birthdate of these neurons correlates with their final position in the adult cortex, resulting in unanticipated specializations of the retinotopic circuit in the anterior-posterior axis of the visual system. The concurrent temporal patterning of symmetrically and asymmetrically dividing neural stem cells thus acts as a powerful mechanism to couple the generation of neural diversity with circuit patterning.