bluuebunny/crossref_metadata_2025 huggingface dataset:
Strong Genetic Overlaps Between Dimensional and Categorical Models of Bipolar Disorders in a Family Sample
Authors: Arbona-Lampaya Alejandro , Sung Heejong , D’Amico Alexander , Knowles Emma E. M. , Besançon Emily K. , Freifeld Ally , Lacbawan Ley , Lopes Fabiana , Kassem Layla , Nardi Antonio E. , McMahon Francis J.
Published: June 2023

Background

Bipolar disorder (BD) presents with a wide range of symptoms that vary among relatives, casting doubt on categorical illness models. To address this uncertainly, we investigated the heritability and genetic relationships between categorical and dimensional models of BD in a family sample.

Methods

Participants in the Amish-Mennonite Bipolar Genetics (AMBiGen) study were assigned categorical mood disorder diagnoses by structured psychiatric interview and completed the Mood Disorder Questionnaire (MDQ), which assesses lifetime history of manic symptoms and associated impairment. Major MDQ dimensions were analyzed by Principal Component Analysis (PCA) in 726 participants. Heritability and genetic overlaps between categorical diagnoses and MDQ-derived dimensions were estimated with SOLAR-ECLIPSE within 432 genotyped participants.

Results

MDQ scores were significantly higher among individuals diagnosed with BD and related disorders, as expected, but varied widely among relatives. PCA suggested a three-component model for the MDQ. Heritability of the MDQ score was 30% (p<0.001), evenly distributed across its three principal components. Strong and significant genetic correlations were found between categorical diagnoses and most MDQ measures.

Limitations

Recruitment through probands with BD resulted in increased prevalence of BD in this sample, limiting generalizability. Unavailable genetic data reduced sample size for some analyses.

Conclusion

Significant heritability and high genetic correlations between categorical diagnoses and MDQ measures support a genetic continuity between dimensional and categorical models of BD.
Repeat investigation during social preference behavior is suppressed in male mice with prefrontal cortexcacna1c(Cav1.2)-deficiency through the dysregulation of neural dynamics
Authors: Hackett Jonathan , Nadkarni Viraj , Singh Ronak S. , Carthy Camille L. , Antigua Susan , Hall Baila S. , Rajadhyaksha Anjali M.
Published: June 2023

Impairments in social behavior are observed in a range of neuropsychiatric disorders and several lines of evidence have demonstrated that dysfunction of the prefrontal cortex (PFC) plays a central role in social deficits. We have previously shown that loss of neuropsychiatric risk geneCacna1cthat codes for the Cav1.2 isoform of L-type calcium channels (LTCCs) in the PFC result in impaired sociability as tested using the three-chamber social approach test. In this study we aimed to further characterize the nature of the social deficit associated with a reduction in PFC Cav1.2 channels (Cav1.2PFCKOmice) by testing male mice in a range of social and non-social tests while examining PFC neural activity usingin vivoGCaMP6s fiber photometry. We found that during the first investigation of the social and non-social stimulus in the three-chamber test, both Cav1.2PFCKOmale mice and Cav1.2PFCGFPcontrols spent significantly more time with the social stimulus compared to a non-social object. In contrast, during repeat investigations while Cav1.2PFCWTmice continued to spend more time with the social stimulus, Cav1.2PFCKOmice spent equal amount of time with both social and non-social stimuli. Neural activity recordings paralleled social behavior with increase in PFC population activity in Cav1.2PFCWTmice during first and repeat investigations, which was predictive of social preference behavior. In Cav1.2PFCKOmice, there was an increase in PFC activity during first social investigation but not during repeat investigations. These behavioral and neural differences were not observed during a reciprocal social interaction test nor during a forced alternation novelty test. To evaluate a potential deficit in reward-related processes, we tested mice in a three-chamber test wherein the social stimulus was replaced by food. Behavioral testing revealed that both Cav1.2PFCWTand Cav1.2PFCKOmice showed a preference for food over object with significantly greater preference during repeat investigation. Interestingly, there was no increase in PFC activity when Cav1.2PFCWTor Cav1.2PFCKOfirst investigated the food however activity significantly increased in Cav1.2PFCWTmice during repeat investigations of the food. This was not observed in Cav1.2PFCKOmice. In summary, a reduction in Cav1.2 channels in the PFC suppresses the development of a sustained social preference in mice that is associated with lack of PFC neuronal population activity that may be related to deficits in social reward.
KRAB-zinc-finger proteins regulate endogenous retroviruses to sculpt germline transcriptomes and genome evolution
Authors: Otsuka Kai , Sakashita Akihiko , Maezawa So , Schultz Richard M. , Namekawa Satoshi H.
Published: June 2023

As transposable elements (TEs) coevolved with the host genome, the host genome exploited TEs as functional regulatory elements. What remains largely unknown are how the activity of TEs, namely, endogenous retroviruses (ERVs), are regulated and how TEs evolved in the germline. Here we show that KRAB domain-containing zinc-finger proteins (KZFPs), which are highly expressed in mitotically dividing spermatogonia, bind to suppressed ERVs that function following entry into meiosis as active enhancers. These features are observed for independently evolved KZFPs and ERVs in mice and humans, i.e., are evolutionarily conserved in mammals. Further, we show that meiotic sex chromosome inactivation (MSCI) antagonizes the coevolution of KZFPs and ERVs in mammals. Our study uncovers a mechanism by which KZFPs regulate ERVs to sculpt germline transcriptomes. We propose that epigenetic programming in the mammalian germline during the mitosis-to-meiosis transition facilitates coevolution of KZFPs and TEs on autosomes and is antagonized by MSCI.
Multi-axial Profile Scores (MAPS) for Complex Child Psychiatric Presentations
Authors: Adams Michelle , Tonge Bruce , Pantelis Christos , Merritt Antonia , Moseley David , De Netto Riana , Syeda Warda , Wannan Cassandra
Published: June 2023

The authors have withdrawn their manuscript owing to it needing further revision before publication, as well as errors in the author list. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
An integrated mechanistic and data-driven computational model predicts cell responses to high- and low-affinity EGFR ligands
Authors: Myers Paul J. , Lee Sung Hyun , Lazzara Matthew J.
Published: June 2023

The biophysical properties of ligand binding heavily influence the ability of receptors to specify cell fates. Understanding the rules by which ligand binding kinetics impact cell phenotype is challenging, however, because of the coupled information transfers that occur from receptors to downstream signaling effectors and from effectors to phenotypes. Here, we address that issue by developing an integrated mechanistic and data-driven computational modeling platform to predict cell responses to different ligands for the epidermal growth factor receptor (EGFR). Experimental data for model training and validation were generated using MCF7 human breast cancer cells treated with the high- and low-affinity ligands epidermal growth factor (EGF) and epiregulin (EREG), respectively. The integrated model captures the unintuitive, concentration-dependent abilities of EGF and EREG to drive signals and phenotypes differently, even at similar levels of receptor occupancy. For example, the model correctly predicts the dominance of EREG over EGF in driving a cell differentiation phenotype through AKT signaling at intermediate and saturating ligand concentrations and the ability of EGF and EREG to drive a broadly concentration-sensitive migration phenotype through cooperative ERK and AKT signaling. Parameter sensitivity analysis identifies EGFR endocytosis, which is differentially regulated by EGF and EREG, as one of the most important determinants of the alternative phenotypes driven by different ligands. The integrated model provides a new platform to predict how phenotypes are controlled by the earliest biophysical rate processes in signal transduction and may eventually be leveraged to understand receptor signaling system performance depends on cell context.

One-sentence summary

Integrated kinetic and data-driven EGFR signaling model identifies the specific signaling mechanisms that dictate cell responses to EGFR activation by different ligands.
Neural tube organoids self-organise floorplate through BMP-mediated cluster competition
Authors: Krammer Teresa , Stuart Hannah T. , Gromberg Elena , Ishihara Keisuke , Melchionda Manuela , Wang Jingkui , Costantini Elena , Lehr Stefanie , Cislo Dillon , Arbanas Laura , Hörmann Alexandra , Neumüller Ralph A. , Elvassore Nicola , Siggia Eric , Briscoe James , Kicheva Anna , Tanaka Elly M.
Published: June 2023

The neural tube (NT) has been a hallmark example of embryonic induction and patterning whereby the notochord induces an organiser, the floorplate, that secretes Sonic Hedgehog (SHH) to pattern the surrounding field of neural progenitors. On the other hand, NT organoids (NTOs) formed from embryonic stem cells (ESCs) undergo spontaneous floorplate formation and patterning in the absence of their normal embryonic inducers. Understanding how stem cells undergo regulative organiser formation is a central challenge in biology. Here, we investigated the self-organisation of a SHH-expressing floorplate organiser using clonal NTOs. Expression of FOXA2, a floorplate transcription factor, was initially spatially scattered before resolving into multiple clusters. These FOXA2+clusters underwent competition and physical sorting, resulting in a stable “winning” floorplate. We identified BMP signalling as a key governor of long-range cluster competition. FOXA2+clusters expressed BMP4 ligand suppressing FOXA2 in receiving cells, while simultaneously expressing the BMP-inhibitor NOGGIN to secure FOXA2+cluster survival. Genetic mutation ofNogginperturbed the floorplate not only in NTOs but alsoin vivoat the mid-hindbrain region of the mouse NT. These results demonstrate how the floorplate can form autonomously without its well-known inducer, the notochord, suggesting redundant mechanisms ensuring robustness. Defining molecular pathways that govern organiser self-organisation is critical in harnessing the developmental plasticity of stem cells toward directed tissue engineering.
Small allelic variants are a source of ancestral bias in structural variant breakpoint placement
Authors: Audano Peter A. , Beck Christine R.
Published: June 2023

High-quality genome assemblies and sophisticated algorithms have increased sensitivity for a wide range of variant types, and breakpoint accuracy for structural variants (SVs, ≥ 50 bp) has improved to near basepair precision. Despite these advances, many SVs in unique regions of the genome are subject to systematic bias that affects breakpoint location. This ambiguity leads to less accurate variant comparisons across samples, and it obscures true breakpoint features needed for mechanistic inferences. To understand why SVs are not consistently placed, we re-analyzed 64 phased haplotypes constructed from long-read assemblies released by the Human Genome Structural Variation Consortium (HGSVC). We identified variable breakpoints for 882 SV insertions and 180 SV deletions not anchored in tandem repeats (TRs) or segmental duplications (SDs). While this is unexpectedly high for genome assemblies in unique loci, we find read-based callsets from the same sequencing data yielded 1,566 insertions and 986 deletions with inconsistent breakpoints also not anchored in TRs or SDs. When we investigated causes for breakpoint inaccuracy, we found sequence and assembly errors had minimal impact, but we observed a strong effect of ancestry. We confirmed that polymorphic mismatches and small indels are enriched at shifted breakpoints and that these polymorphisms are generally lost when breakpoints shift. Long tracts of homology, such as SVs mediated by transposable elements, increase the likelihood of imprecise SV calls and the distance they are shifted. Tandem Duplication (TD) breakpoints are the most heavily affected SV class with 14% of TDs placed at different locations across haplotypes. While graph genome methods normalize SV calls across many samples, the resulting breakpoints are sometimes incorrect, highlighting a need to tune graph methods for breakpoint accuracy. The breakpoint inconsistencies we characterize collectively affect ∼5% of the SVs called in a human genome and underscore a need for algorithm development to improve SV databases, mitigate the impact of ancestry on breakpoint placement, and increase the value of callsets for investigating mutational processes.
Rapid and accurate multi-phenotype imputation for millions of individuals
Authors: Gu Lin-Lin , Wu Hong-Shan , Liu Tian-Yi , Zhang Yong-Jie , He Jing-Cheng , Liu Xiao-Lei , Wang Zhi-Yong , Chen Guo-Bo , Jiang Dan , Fang Ming
Published: June 2023

Deep phenotyping can enhance the power of genetic analysis, including genome-wide association studies (GWAS), but the occurrence of missing phenotypes compromises the potential of such resources. Although many phenotypic imputation methods have been developed, the accurate imputation of millions of individuals remains extremely challenging. In the present study, we developed a novel multi-phenotype imputation method based on mixed fast random forest (PIXANT) by leveraging efficient machine learning (ML)-based algorithms. We demonstrate that PIXANT runtime is faster and computer memory usage is less than that of other state-of-the-art methods when applied to the UK Biobank (UKB) data, suggesting that PIXANT is scalable to cohorts with millions of individuals. Our simulations with hundreds of individuals showed that PIXANT accuracy was superior to or comparable to the accuracy of the most advanced methods available. PIXANT was used to impute 425 phenotypes for the UKB data of 277,301 unrelated White British citizens. When GWAS was subsequently performed on the imputed phenotypes, 18.4% more GWAS loci were identified than before imputation (8,710 vs 7,355). The increased statistical power of GWAS identified novel positional candidate genes affecting heart rate, such as RNF220, SCN10A, and RGS6, suggesting that the use of imputed phenotype data from a large cohort may lead to the discovery of novel genes for complex traits.
Robust estimation of brain stimulation evoked responses using magnetoencephalography
Authors: Oswal Ashwini , Abdi-Sargezeh Bahman , Özkurt Tolga Esat , Taulu Samu , Sarangmat Nagaraja , Green Alexander L , Litvak Vladimir
Published: June 2023

Magnetoencephalography (MEG) recordings are often contaminated by interference that can exceed the amplitude of physiological brain activity by several orders of magnitude. Furthermore, activity of interference sources may‘leak’into the activity of brain signals of interest, resulting in source estimation inaccuracies. This problem is particularly apparent when using MEG to interrogate the effects of brain stimulation on large scale cortical networks.

This technical report offers two contributions. Firstly, using phantom MEG recordings we describe an approach for validating the estimation accuracy of brain stimulation evoked responses. Secondly, we propose a novel denoising method for suppressing the leakage of stimulation related signal into recorded brain activity. This approach leverages spatial and temporal domain projectors for signal arising from prespecified anatomical regions of interest. We highlight its advantages compared to the benchmark - spatiotemporal signal space separation (tSSS) - and show that it can more accurately reveal brain stimulation evoked responses.
Two independent DNA repair pathways cause mutagenesis in template switching deficientSaccharomyces cerevisiae
Authors: Jiang Yangyang Kate , Medley Eleanor , Brown Grant W.
Published: June 2023

Upon DNA replication stress, cells utilize the post-replication repair pathway to repair single-stranded DNA and maintain genome integrity. Post-replication repair is divided into two branches: error-prone translesion synthesis, signaled by PCNA mono-ubiquitination, and error-free template switching, signaled by PCNA poly-ubiquitination. InSaccharomyces cerevisiae, Rad5 is involved in both branches of repair during DNA replication stress. When the PCNA poly-ubiquitination function of Rad5 is disrupted, Rad5 recruits translesion synthesis polymerases to stalled replication forks, resulting in mutagenic repair. Details of how mutagenic repair is carried out, as well as the relationship between Rad5-mediated mutagenic repair and the canonical PCNA-mediated mutagenic repair, remain to be understood. We find that Rad5-mediated mutagenic repair requires the translesion synthesis polymerase ζ but does not require other yeast translesion polymerase activities. Furthermore, we show that Rad5-mediated mutagenic repair is independent of PCNA binding by Rev1 and so is separable from canonical mutagenic repair. In the absence of error-free template switching, both modes of mutagenic repair contribute additively to replication stress response in a replication timing-independent manner. Cellular contexts where error-free template switching is compromised are not simply laboratory phenomena, as we find that a natural variant inRAD5is defective in PCNA poly-ubiquitination and therefore defective in error-free repair, resulting in Rad5- and PCNA-mediated mutagenic repair. Our results highlight the importance of Rad5 in regulating spontaneous mutagenesis and genetic diversity inS. cerevisiaethrough different modes of post-replication repair.
CaBLAM! A high-contrast bioluminescent Ca2+indicator derived from an engineeredOplophorus gracilirostrisluciferase
Authors: Lambert Gerard G. , Crespo Emmanuel L. , Murphy Jeremy , Boassa Daniela , Luong Selena , Celinskis Dmitrijs , Venn Stephanie , Nguyen Daniel K. , Hu Junru , Sprecher Brittany , Tree Maya O. , Orcutt Richard , Heydari Daniel , Bell Aidan B. , Torreblanca-Zanca Albertina , Hakimi Ali , Lipscombe Diane , Moore Christopher I. , Hochgeschwender Ute , Shaner Nathan C.
Published: June 2023

Ca2+plays many critical roles in cell physiology and biochemistry, leading researchers to develop a number of fluorescent small molecule dyes and genetically encodable probes that optically report changes in Ca2+concentrations in living cells. Though such fluorescence-based genetically encoded Ca2+indicators (GECIs) have become a mainstay of modern Ca2+sensing and imaging, bioluminescence-based GECIs—probes that generate light through oxidation of a small-molecule by a luciferase or photoprotein—have several distinct advantages over their fluorescent counterparts. Bioluminescent tags do not photobleach, do not suffer from nonspecific autofluorescent background, and do not lead to phototoxicity since they do not require the extremely bright extrinsic excitation light typically required for fluorescence imaging, especially with 2-photon microscopy. Current BL GECIs perform poorly relative to fluorescent GECIs, producing small changes in bioluminescence intensity due to high baseline signal at resting Ca2+concentrations and suboptimal Ca2+affinities. Here, we describe the development of a new bioluminescent GECI, “CaBLAM,” which displays much higher contrast (dynamic range) than previously described bioluminescent GECIs and has a Ca2+affinity suitable for capturing physiological changes in cytosolic Ca2+concentration. Derived from a new variant ofOplophorus gracilirostrisluciferase with superior in vitro properties and a highly favorable scaffold for insertion of sensor domains, CaBLAM allows for single-cell and subcellular resolution imaging of Ca2+dynamics at high frame rates in cultured neurons andin vivo. CaBLAM marks a significant milestone in the GECI timeline, enabling Ca2+recordings with high spatial and temporal resolution without perturbing cells with intense excitation light.
History of incarceration and age-related cognitive impairment: Testing models of genetic and environmental risk in longitudinal panel study of older adults
Authors: Tanksley Peter T. , Logan Matthew W. , Barnes J.C.
Published: June 2023

History of incarceration is associated with an excess of morbidity and mortality. While the incarceration experience itself comes with substantive health risks (e.g., injury, psychological stress, exposure to infectious disease), most inmates eventually return to the general population where they will be diagnosed with the same age-related conditions that drive mortality in the non-incarcerated population but at exaggerated rates. However, the interplay between history of incarceration as a risk factor and more traditional risk factors for age-related diseases (e.g., genetic risk factors) has not been studied. Here, we focus on cognitive impairment, a hallmark of neurodegenerative conditions like Alzheimer’s disease, as an age-related state that may be uniquely impacted by the confluence of environmental stressors (e.g., incarceration) and genetic risk factors. Using data from the Health and Retirement Study, we found that incarceration andAPOE-ε4genotype (i.e., the chief genetic risk factor for Alzheimer’s disease) both constituted substantive risk factors for cognitive impairment in terms of overall risk and earlier onset. The observed effects were mutually independent, however, suggesting that the risk conveyed by incarceration andAPOE-ε4genotype operate across different risk pathways. Our results have implications for the study of criminal justice contact as a public health risk factor for age-related, neurodegenerative conditions.
Generalizable and Automated Classification of TNM Stage from Pathology Reports with External Validation
Authors: Kefeli Jenna , Tatonetti Nicholas
Published: June 2023

Cancer staging is an essential clinical attribute informing patient prognosis and clinical trial eligibility. However, it is not routinely recorded in structured electronic health records. Here, we present a generalizable method for the automated classification of TNM stage directly from pathology report text. We train a BERT-based model using publicly available pathology reports across approximately 7,000 patients and 23 cancer types. We explore the use of different model types, with differing input sizes, parameters, and model architectures. Our final model goes beyond term-extraction, inferring TNM stage from context when it is not included in the report text explicitly. As external validation, we test our model on almost 8,000 pathology reports from Columbia University Medical Center, finding that our trained model achieved an AU-ROC of 0.815-0.942. This suggests that our model can be applied broadly to other institutions without additional institution-specific fine-tuning.
Prevalence and correlates of Metabolic Syndrome among adults in Freetown, Sierra Leone: A comparative analysis of NCEP ATP III, IDF and Harmonized ATP III criteria
Authors: Russell James Baligeh Walter , Koroma Theresa Ruba , Sesay Santigie , Samura Sallieu K , Lakoh Sulaiman , Bockarie Ansumana , Abiri Onomeh Thomas , Conteh Victor , Conteh Sorie , Smith Mohamed , Mahdi O Z , Lisk Durodami. R.
Published: June 2023

Background

Metabolic syndrome (MS) is a global health concern, especially for low and middle-income countries with limited resources and information. The study’s objective was to assess the prevalence of MS in Freetown, Sierra Leone, using the Adult Treatment Panel III (ATP III), International Diabetes Federation (IDF) and Harmonize ATP III. Additionally, we aimed to establish the concordance between these three different criteria used.

Methods

This community-based health screening survey was conducted from October 2019 to October 2022. A multistage stratified random design was used to select adults aged 20 years and above. Mean, interquartile range (IQR), and logistic regression were used for statistical analysis. The kappa coefficient statistics resolved the agreement between these defined criteria.

Results

The prevalence for NCEP ATP III, Harmonize ATP III and IDF criteria was 11.8% (95% CI: 9.0 - 15.15), 14.3% (95% CI: 11.3 - 18.0), and 8.5% (95% CI: 6.2 - 11.2), respectively for the 2,394 selected adults. The kappa coefficient (κ) agreement between the MS is: Harmonized ATP III and IDF criteria = [(208 (60.8%); (κ =0.62)]; Harmonized ATP III and NCEP ATP III = [(201 (58.7%); (κ =0.71)]; while IDF and NCEP ATP III was [(132 (38.6%); (κ =0.52)]. In the multivariable regression analysis, waist circumference correlated with all three MS criteria: ATP III [AOR = 0.85; C.I 95%: (0.40-1.78), p = 0.032], Harmonized ATP III [AOR = 1.14; C.I 95%: (0.62-2.11), p = 0.024], IDF [AOR = 1.06; C.I 95% (0.52-2.16), p = 0.018]

Conclusion

We reported a high prevalence of MS in Freetown, Sierra Leone and identified waist circumference as a major risk factor for MS. This underscores the crucial role of health education and effective management of MS in Sierra Leone.
HD mutation results in a dominant negative effect on HTT function
Authors: Laundos Tiago L. , Li Shu , Cheang Eric , Santis Riccardo De , Piccolo Francesco M. , Brivanlou Ali H.
Published: June 2023

Huntington’s disease (HD) remains an incurable and fatal neurodegenerative disease long after CAG-expansion mutation in the huntingtin gene (HTT) was identified as the cause. The underlying pathological mechanism, whether HTT loss of function or gain of toxicity results from mutation, remains a matter of debate. In this study, we genetically modulated wild-type or mutant HTT expression levels in isogenic human embryonic stem cells to systematically investigate their contribution to HD-specific phenotypes. Using highly reproducible and quantifiablein vitromicropattern-based assays, we observed comparable phenotypes with HD mutation and HTT depletion. However, halving endogenous wild-type HTT levels did not strongly recapitulate the HD phenotypes, arguing against a classical loss of function mechanism. Remarkably, expression of CAG-expanded HTT in non-HD cells induced HD-like phenotypes akin to HTT depletion. By corollary, these results indicate a dominant negative effect of mutated HTT on its wild-type counterpart. Complementation with additional copies of wild-type HTT ameliorated the HD-associated phenotypes, strongly supporting a classical dominant negative mechanism. Understanding the molecular basis of this dominant negative effect will guide the development of efficient clinical strategies to counteract the deleterious impact of mutant HTT on the wild-type protein.
Revisiting the role of cAMP in Drosophila aversive olfactory memory formation
Authors: Abe Takashi , Yamazaki Daisuke , Hiroi Makoto , Ueoka Yutaro , Maeyama Yuko , Tabata Tetsuya
Published: June 2023

In the olfactory aversive conditioning of Drosophila melanogaster, an odor, the conditioned stimulus (CS), is associated with electric shock, the unconditioned stimulus (US). The Rutabaga adenylyl cyclase in Kenyon cells (KCs) of the fly brain synthesizes cAMP, which is believed to serve as the coincidence detector synergistically stimulated by calcium/calmodulin evoked by the odor reception and GαS released in response to the dopamine signaling elicited by electric shock. However, live imaging analyses revealed that olfactory stimulation itself prompted the activation of dopaminergic neurons and resulted in the elevation of cAMP levels in KCs that received dopamine, regardless of calcium signaling. This finding raises questions about the longstanding and fundamental comprehension of conditioning mechanisms, as the cAMP levels in conditioned stimulus-positive (CS+) KCs could not be distinguished from those in the rest of the KCs. Our findings suggest that cAMP concentrations do not function as a mnemonic engram to CS+ neurons. Rather, the collective dynamics of cAMP are capable of integratively encoding the valence ascribed to odors being perceived at a given time. Furthermore, our investigation revealed that associative conditioning induces modifications in dopamine levels in response to both CS and US, resulting in alterations in cAMP levels. The increase in cAMP exerts a deleterious effect on the acetylcholine transmission from KCs. Accordingly, we postulate that, during conditioning, cAMP depresses KCs, thereby skewing the valence of the CS+ odor towards a more aversive state for the conditioned flies, and ultimately culminating in the formation of aversive memories.
Synaptic vesicle glycoprotein 2C enhances vesicular storage of dopamine and counters dopaminergic toxicity
Authors: Bucher Meghan L , Dunn Amy R , Bradner Joshua M , Egerton Kristen Stout , Burkett James P , Johnson Michelle A , Miller Gary W
Published: June 2023

Dopaminergic neurons of the substantia nigra exist in a persistent state of vulnerability resulting from high baseline oxidative stress, high energy demand, and broad unmyelinated axonal arborizations. Impairments in the storage of dopamine compound this stress due to cytosolic reactions that transform the vital neurotransmitter into an endogenous neurotoxicant, and this toxicity is thought to contribute to the dopamine neuron degeneration that occurs Parkinson’s disease. We have previously identified synaptic vesicle glycoprotein 2C (SV2C) as a modifier of vesicular dopamine function, demonstrating that genetic ablation of SV2C in mice results in decreased dopamine content and evoked dopamine release in the striatum. Here, we adapted a previously publishedin vitroassay utilizing false fluorescent neurotransmitter 206 (FFN206) to visualize how SV2C regulates vesicular dopamine dynamics and determined that SV2C promotes the uptake and retention of FFN206 within vesicles. In addition, we present data indicating that SV2C enhances the retention of dopamine in the vesicular compartment with radiolabeled dopamine in vesicles isolated from immortalized cells and from mouse brain. Further, we demonstrate that SV2C enhances the ability of vesicles to store the neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) and that genetic ablation of SV2C results in enhanced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced vulnerability in mice. Together, these findings suggest that SV2C functions to enhance vesicular storage of dopamine and neurotoxicants, and helps maintain the integrity of dopaminergic neurons.
“In vitro construction and long read sequencing analysis of a 24 kb long artificial DNA sequence encoding the Universal Declaration of the Rights of Man and of the Citizen ”
Authors: Leblanc Julien , Boulle Olivier , Roux Emeline , Nicolas Jacques , Lavenier Dominique , Audic Yann
Published: June 2023

In absence of DNA template, theab initioproduction of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. We propose in this report a fullyin vitroprotocol to generate very long double-stranded DNA molecule starting from commercially available short DNA blocks in less than 3 days. This innovative application of Golden Gate assembly allowed us to streamline the assembly process to produce a 24 kb long DNA molecule storing part of the Universal Declaration of Human rights and citizens. The DNA molecule produced can be readily cloned into suitable host/vector system for amplification and selection.
The mechanism of Atg15-mediated membrane disruption in autophagy
Authors: Kagohashi Yoko , Sasaki Michiko , May Alexander I , Kawamata Tomoko , Ohsumi Yoshinori
Published: June 2023

Autophagy is a lysosomal/vacuolar delivery system that isolates and degrades cytoplasmic material. Following delivery by autophagosomes, cytoplasmic components are released into the vacuole within an autophagic body (AB), which is a single-membrane structure derived from the inner membrane of the autophagosome. This membrane must be disrupted for degradation of the cytoplasmic cargo to occur. The vacuolar proteases Pep4 and Prb1, as well as the lipase Atg15, are known to be necessary for this process, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and use it to show that Atg15 is the sole vacuolar phospholipase and that Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments also reveal that Atg15 is a B-type phospholipase of broad substrate specificity that is likely implicated in the disruption of a range of membranes delivered to the vacuole. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.
Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches
Authors: Procknow Rebecca R. , Kennedy Kristopher J. , Kluba Maxwell , Rodriguez Lesley J. , Taga Michiko E.
Published: June 2023

The ability to sense and respond to intracellular metabolite levels enables cells to adapt to environmental conditions. Many prokaryotes use riboswitches – structured RNA elements usually located in the 5’ untranslated region of mRNAs – to sense intracellular metabolites and respond by modulating gene expression. The corrinoid riboswitch class, which responds to adenosylcobalamin (coenzyme B12) and related metabolites, is among the most widespread in bacteria. The structural elements for corrinoid binding and the requirement for a kissing loop interaction between the aptamer and expression platform domains have been established for several corrinoid riboswitches. However, the conformational changes in the expression platform that modulate gene expression in response to corrinoid binding remain unknown. Here, we employ anin vivoGFP reporter system inBacillus subtilisto define alternative secondary structures in the expression platform of a corrinoid riboswitch fromPriestia megateriumby disrupting and restoring base-pairing interactions. Moreover, we report the discovery and characterization of the first riboswitch known to activate gene expression in response to corrinoids. In both cases, mutually exclusive RNA secondary structures are responsible for promoting or preventing the formation of an intrinsic transcription terminator in response to the corrinoid binding state of the aptamer domain. Knowledge of these regulatory mechanisms allowed us to develop synthetic corrinoid riboswitches that convert repressing riboswitches to riboswitches that robustly induce gene expression in response to corrinoids. Due to their high expression levels, low background, and over 100-fold level of induction, these synthetic riboswitches have potential use as biosensors or genetic tools.
Rapid species discovery and identification with real-time barcoding facilitated by ONTbarcoder 2.0 and Oxford Nanopore R10.4
Authors: Srivathsan Amrita , Feng Vivian , Suárez Daniel , Emerson Brent , Meier Rudolf
Published: June 2023

Most arthropod species are undescribed and hidden in biodiversity samples that are difficult to sort to species using morphological characters. Sorting specimens to putative species with DNA barcodes is an attractive alternative, but needs cost-effective techniques that are suitable for use in many laboratories around the world. Barcoding with Oxford Nanopore’s portable and inexpensive MinION sequencers could be a good presorting approach because it reduces the space and capital cost of a fully functional barcoding laboratory. Similarly important would be user-friendly and reliable software for analysis of the ONT data. It is here provided in the form of an updated ONTbarcoder that is available for all commonly used operating systems and includes a GUI. The new version of ONTbarcoder has three key improvements related to the higher read quality obtained with ONT’s latest basecallers and chemistry (R10.4 flow cells, Q20+ kits). Firstly, ONTbarcoder can now deliver real-time barcoding to complement ONT’s real-time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run and a significant proportion of barcodes are available before sequencing completes. This real-time feature of ONTbarcoder can be configured to handle both Mk1B and Mk1C sequencers. The input is a demultiplexing sheet and sequencing data (raw or basecalled). Secondly, the improved read quality of ONT’s latest flow cells (R10.4) allows for the use of primers with shorter indices than those previously needed (9 bp vs. 12-13 bp). This decreases primer cost and improves PCR success rates. Thirdly, we demonstrate that the availability of R10.4 chemistry in the low-cost Flongle flow cell is an attractive option for users who require only 200-250 barcodes at a time.
Endosomal dysfunction contributes to cerebellar deficits in spinocerebellar ataxia type 6
Authors: Cook Anna A. , Leung Tsz Chui Sophia , Rice Max , Nachman Maya , Zadigue-Dubé Élyse , Watt Alanna J.
Published: June 2023

Spinocerebellar ataxia type 6 (SCA6) is a rare disease that is characterized by cerebellar dysfunction. Patients have progressive motor coordination impairment, and postmortem brain tissue reveals degeneration of cerebellar Purkinje cells and a reduced level of cerebellar brain-derived neurotrophic factor (BDNF). However, the pathophysiological changes underlying SCA6 are not fully understood. We carried out RNA sequencing of cerebellar vermis tissue in a mouse model of SCA6, which revealed widespread dysregulation of genes associated with the endo-lysosomal system. Since disruption to endosomes or lysosomes could contribute to cellular deficits, we examined the endo-lysosomal system in SCA6. We identified alterations in multiple endosomal compartments in the Purkinje cells of SCA6 mice. Early endosomes were enlarged, while the size of the late endosome compartment was reduced. We also found evidence for impaired trafficking of cargo to the lysosomes. As the proper functioning of the endo-lysosomal system is crucial for the sorting and trafficking of signaling molecules, we wondered whether these changes could contribute to previously identified deficits in signaling by BDNF and its receptor tropomyosin kinase B (TrkB) in SCA6. Indeed, we found that the enlarged early endosomes in SCA6 mice accumulated both BDNF and TrkB. Furthermore, TrkB recycling to the cell membrane in recycling endosomes was reduced, and the late endosome transport of BDNF for degradation was impaired. Therefore, mis-trafficking due to aberrant endo-lysosomal transport and function could contribute to SCA6 pathophysiology through alterations to BDNF-TrkB signaling, as well as mishandling of other signaling molecules. Deficits in early endosomes and BDNF localization were rescued by chronic administration of a TrkB agonist, 7,8-DHF, that we have previously shown restores motor coordination and cerebellar TrkB expression. The endo-lysosomal system is thus both a novel locus of pathophysiology in SCA6, and a promising therapeutic target.
Cortical over-representation of phonetic onsets of ignored speech in hearing impaired individuals
Authors: Carta Sara , Aličković Emina , Zaar Johannes , López Valdes Alejandro , Di Liberto Giovanni M.
Published: June 2023

Hearing impairment alters the sound input received by the human auditory system, reducing speech comprehension in noisy multi-talker auditory scenes. Despite such challenges, attentional modulation on the envelope tracking in multi-talker scenarios is comparable between normal hearing (NH) and hearing impaired (HI) participants, with previous research suggesting an over-representation of the speech envelopes in HI individuals (see, e.g., Fuglsang et al. 2020 and Presacco et al. 2019), even though HI participants reported difficulties in performing the task. This result raises an important question: What speech-processing stage could reflect the difficulty in attentional selection, if not envelope tracking? Here, we use scalp electroencephalography (EEG) to test the hypothesis that such difficulties are underpinned by an over-representation of phonological-level information of the ignored speech sounds. To do so, we carried out a re-analysis of an EEG dataset where EEG signals were recorded as HI participants fitted with hearing aids attended to one speaker (target) while ignoring a competing speaker (masker) and spatialised multi-talker background noise. Multivariate temporal response function analyses revealed that EEG signals reflect stronger phonetic-feature encoding for target than masker speech streams. Interestingly, robust EEG encoding of phoneme onsets emerged for both target and masker streams, in contrast with previous work on NH participants and in line with our hypothesis of an over-representation of the masker. Stronger phoneme-onset encoding emerged for the masker, pointing to a possible neural basis for the higher distractibility experienced by HI individuals.

Significance Statement

This study investigated the neural underpinnings of attentional selection in multi-talker scenarios in hearing-impaired participants. The impact of attentional selection on phonological encoding was assessed with electroencephalography (EEG) in an immersive multi-talker scenario. EEG signals encoded the phonetic features of the target (attended) speech more strongly than those of the masker (ignored) speech; but interestingly, they encoded the phoneme onsets of both target and masker speech. This suggests that the cortex of hearing-impaired individuals may over-represent higher-level features of ignored speech sounds, which could contribute to their higher distractibility in noisy environments. These findings provide insight into the neural mechanisms underlying speech comprehension in hearing-impaired individuals and could inform the development of novel approaches to improve speech perception in noisy environments.
Unmasking Crucial Residues in Adipose Triglyceride Lipase (ATGL) for Co-Activation with Comparative Gene Identification-58 (CGI-58)
Authors: Kulminskaya Natalia , Rodriguez Gamez Carlos Francisco , Hofer Peter , Cerk Ines Kathrin , Dubey Noopur , Viertlmayr Roland , Sagmeister Theo , Pavkov-Keller Tea , Zechner Rudolf , Oberer Monika
Published: June 2023

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be co-activated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein co-activation on a per-residue level. We identified the region from residues N209-N215 in mouse ATGL as essential for co-activation by mouse CGI-58. ATGL variants with amino-acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 co-activation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the co-activation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for co-activation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58 mediated co-activation of ATGL.
Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism
Authors: Pujadas Emily M. , Wei Xiaolong , Acosta Nicolas , Carter Lucas , Yang Jiekun , Almassalha Luay , Daneshkhah Ali , Rao Suhas S.P. , Agrawal Vasundhara , Seker-Polat Fidan , Aiden Erez Lieberman , Kanemaki Masato T. , Backman Vadim , Adli Mazhar
Published: June 2023

BACKGROUND

B-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology.

RESULTS

Paired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy,in situHi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning.

CONCLUSIONS

Our findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.
Differential regulation of hair cell actin cytoskeleton mediated by SRF and MRTFB
Authors: Zhou Ling-Yun , Jin Chen-Xi , Wang Wen-Xiao , Song Lei , Shin Jung-Bum , Du Ting-Ting , Wu Hao
Published: June 2023

The MRTF-SRF pathway has been extensively studied for its crucial role in driving the expression of a large number of genes involved in actin cytoskeleton of various cell types. However, the specific contribution of MRTF-SRF in hair cells remains unknown. In this study, we showed that hair cell-specific deletion ofSrforMrtfb, but notMrtfa, leads to similar defects in the development of stereocilia dimensions and the maintenance of cuticular plate integrity. We used FACS-based hair cell RNA-seq analysis to investigate the mechanistic underpinnings of the changes observed inSrfandMrtfbmutants, respectively. Interestingly, the transcriptome analysis revealed distinct profiles of genes regulated bySrfandMrtfb, suggesting different transcriptional regulation mechanisms of actin cytoskeleton activities mediated bySrfandMrtfb. Exogenous delivery of calponin 2 using Adeno-associated virus transduction inSrfmutants partially rescued the impairments of stereocilia dimensions and the F-actin intensity of cuticular plate, suggesting the involvement ofCnn2, as anSrfdownstream target, in regulating the hair bundle morphology and cuticular plate actin cytoskeleton organization. Our study uncovers, for the first time, the unexpected differential transcriptional regulation of actin cytoskeleton mediated bySrfandMrtfbin hair cells, and also demonstrates the critical role of SRF-CNN2 in modulating actin dynamics of the stereocilia and cuticular plate, providing new insights into the molecular mechanism underlying hair cell development and maintenance.