bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulating effects of FGF12 variants on NaV1.2 and NaV1.6 associated with Developmental and Epileptic Encephalopathy and Autism Spectrum Disorder
Authors: Seiffert Simone , Pendziwiat Manuela , Bierhals Tatjana , Goel Himanshu , Schwarz Niklas , van der Ven Amelie , Boßelmann Christian Malte , Willemsen Marjolein H. , Hedrich Ulrike B.S. , Helbig Ingo , Weber Yvonne G.
Published: December 2021

Objective

Fibroblast growth factor 12 (FGF12) may represent an important modulator of neuronal network activity and has been associated with developmental and epileptic encephalopathy (DEE). We sought to identify the underlying pathomechanism of FGF12-related disorders.

Methods

Patients with pathogenic variants in FGF12 were identified through published case reports, GeneMatcher and whole exome sequencing of own case collections. The functional consequences of two missense variants and two copy number variants (CNVs) were studied by co-expression of wild-type and mutant FGF12 in neuronal-like cells (ND7/23) with the sodium channels NaV1.2 or NaV1.6, including their functional active beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B).

Results

Four variants in FGF12 were identified for functional analysis: one novel FGF12 variant in a patient with autism spectrum disorder and three variants from previously published patients affected by developmental and epileptic encephalopathy (DEE). We demonstrate the differential regulating effects of wildtype and mutant FGF12 on NaV1.2 and NaV1.6 channels. Here, FGF12 variants lead to a complex kinetic influence on Nav1.2 and Nav 1.6, including loss- as well as gain-of function changes in fast inactivation as well as loss-of function changes in slow inactivation.

Interpretation

For the first time, we could demonstrate the detailed regulating effect of FGF12 on NaV1.2 and NaV1.6 and confirmed the complex effect of FGF12 on neuronal network activity. Our findings expand the phenotypic spectrum related to FGF12 variants and elucidate the underlying pathomechanism. Specific variants in FGF12-associated disorders may be amenable to precision treatment with sodium channel blockers.
SlTDP1 Is Required To Specify Tapetum Identity And For The Regulation Of Redox Homeostasis In Tomato Anthers
Authors: Salazar-Sarasua Blanca , López-Martín María Jesús , Roque Edelín , Hamza Rim , Cañas Luis Antonio , Beltrán José Pío , Gómez-Mena Concepción
Published: December 2021

The tapetum is a specialized layer of cells within the anther adjacent to the sporogenic tissue. During its short life, it provides nutrients, molecules and materials to the pollen mother cells and microsporocytes being essential during callose degradation and pollen wall formation. However, the acquisition of tapetal cell identity in tomato plants is a process still poorly understood. We report here the identification and characterization of SlTPD1 (Solanum lycopersicum TPD1), a gene specifically required for pollen development in tomato plants. Gene editing was used to generate loss-of-function Sltpd1 mutants that showed absence of tapetal tissue. In these plants, sporogenous cells developed but failed to complete meiosis resulting in complete male sterility. Transcriptomic analysis conducted in wild-type and mutant anthers at an early stage revealed the down regulation of a set of genes related to redox homeostasis. Indeed, Sltpd1 anthers showed a reduction of reactive oxygen species (ROS) accumulation at early stages and altered activity of ROS scavenging enzymes. The obtained results highlight the importance of ROS homeostasis in the interaction between the tapetum and the sporogenous tissue in tomato plants.

One sentence summary

The small protein SlTPD1 is required for tapetum formation in tomato, highlighting the role of this tissue in the regulation of redox homeostasis during male gametogenesis.
Endogenous Syngap1 Alpha Splice Forms Promote Cognitive Function and Seizure Protection
Authors: Kilinc Murat , Arora Vineet , Creson Thomas K. , Rojas Camilo , Le Aliza A. , Lauterborn Julie , Wilkinson Brent , Hartel Nicolas , Graham Nicholas , Reich Adrian , Gou Gemma , Araki Yoichi , Bayés Àlex , Coba Marcelo P. , Lynch Gary , Miller Courtney A. , Rumbaugh Gavin
Published: December 2021

Loss-of-function variants in SYNAGP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-α expression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.
Gene therapy targeting the blood-brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency
Authors: Sundaram Sivaraj M. , Pereira Adriana Arrulo , Köpke Hannes , Müller-Fielitz Helge , De Angelis Meri , Müller Timo D. , Heuer Heike , Körbelin Jakob , Krohn Markus , Mittag Jens , Nogueiras Ruben , Prevot Vincent , Schwaninger Markus
Published: December 2021

The solute carrier monocarboxylate transporter 8 (MCT8) transports the thyroid hormones thyroxine and tri-iodothyronine (T3) across cell membranes. MCT8 gene deficiency, termed Allan-Herndon-Dudley syndrome, is an important cause of X-linked intellectual and motor disability. As no treatment of the neurological symptoms is available yet, we tested a gene replacement therapy in Mct8- and Oatp1c1-deficient mice as a well-established model of the disease. Here, we report that targeting brain endothelial cells for Mct8 expression by intravenously injecting the vector AAV-BR1-Mct8 increased T3 levels in the brain and ameliorated morphological and functional parameters associated with the disease. Importantly, the therapy resulted in a long-lasting improvement in motor coordination. Thus, the data support the concept that MCT8 mediates the transport of thyroid hormones into the brain and indicate that a readily accessible vascular target can help overcome the consequences of the severe disability associated with MCT8 deficiency.
Survival and outcomes of tocilizumab use in severe and critically ill COVID-19 patients not responding to steroids
Authors: Assu Shiraz , Bhasin Deepak , Sekhri Kavita , Sampley Supriya , Singh Harpal , Kaur Gurleen
Published: December 2021

Background

Mortality and morbidity are highest in severe and critically ill patients with COVID -19 pneumonia. We used interleukin-6 (IL-6) receptor inhibitor, tocilizumab in the above patients who failed to show any clinical improvement after initial treatment with steroids.

Methods

This is a retrospective observational study conducted at a tertiary care hospital in India. Severe and critical COVID-19 patients, who got admitted to intensive care unit and subsequently received tocilizumab were included. Patients who worsened clinically or had no change in oxygen requirement even after 24hrs of receiving intravenous methylprednisolone at a dose of 1-2mg/kg/day received a maximum total dose of 800mg of intravenous tocilizumab. The day 28 all-cause mortality and progression to mechanical ventilation were the primary outcome measures. Clinical improvement and oxygen requirements after tocilizumab administration along with trends in inflammatory markers and secondary infections rates were also noted.

Results

A total of 51 patients who did not show clinical improvement even after 24 hours of intravenous steroids had received tocilizumab. In these patients, there was a significant decrease in oxygen requirement and clinical progression by day 7 of tocilizumab administration. Baseline median values of CRP (114.2 mg/L), IL-6 (69.8 pg/ml) and neutrophil-lymphocyte ratio (12.4) in these patients were elevated. Out of these, only CRP showed a significant decrease after the drug administration. 13 (26.5%) of the 49 patients on non-invasive oxygen support progressed to mechanical ventilation and the day 28 all-cause mortality was 10/51(19.6%). 10(19.6%) of the 51 patients had life threatening infections.

Conclusion

Early and timely administration of tocilizumab is a viable option in selected severe and critical COVID-19 patients who do not respond to initial steroids. When given along with steroids, a high suspicion of secondary infections should be kept.
Deviations in Predicted COVID-19 cases in the US during early months of 2021 relate to rise in B.1.526 and its family of variants
Authors: Nandakishore P. , Liu M. , R. Prakash , Gourneni S. , Sukumaran R. , Berman J.M. , Iyer S. , Glorioso C.
Published: December 2021

Objective

To investigate the abrogation of COVID-19 case declines from predicted rates in the US in relationship to viral variants and mutations.

Design

Epidemiological prediction and time series study of COVID-19 in the US by State.

Setting

Community testing and sequencing of COVID-19 in the US.

Participants

Time series US COVID-19 case data from the Johns Hopkins University CSSE database. Time series US Variant and Mutation data from the GISAID database.

Main outcome measures

Primary outcomes were statistical modeling of US state deviations from epidemiological predictions, percentage of COVID-19 variants, percentage of COVID-19 mutations, and reported SARS-CoV-2 infections.

Results

Deviations in epidemiological predictions of COVID-19 case declines in the North Eastern US in March 2021 were highly positively related to percentage of B.1.526 (Iota) lineage (p < 10e − 7) and B.1.526.2 (p < 10 − 8) and the T95I mutation (p < 10e − 9). They were related inversely to B.1.427 and B.1.429 (Epsilon) and there was a trend for association with B.1.1.7 (Alpha) lineage.

Conclusion

Deviations from accurate predictive models are useful for investigating potential immune escape of COVID-19 variants at the population level. The B.1.526 and B.1.526.2 lineages likely have a high potential for immune escape and should be designated as variants of concern. The T95I mutation which is present in the B.1.526, B.1.526.2, and B.1.617.2 (Delta) lineages in the US warrants further investigation as a mutation of concern.
The influence of new SARS-CoV-2 variant Omicron (B.1.1.529) on vaccine efficacy, its correlation to Delta Variants: a computational approach
Authors: Ranjan Prashant , Neha  , Devi Chandra , Devar Kaviyapriya Arulmozhi , Das Parimal
Published: December 2021

The newly discovered COVID variant B.1.1.529 in Botswana has more than 30 mutations in spike and many other in non-spike proteins, far more than any other SARS-CoV-2 variant accepted as a variant of concern by the WHO and officially named Omicron, and has sparked concern among scientists and the general public. Our findings provide insights into structural modification caused by the mutations in the Omicrons receptor-binding domain and look into the effects on interaction with the hosts neutralising antibodies CR3022, B38, CB6, P2B-2F6, and REGN, as well as ACE2R using an in silico approach. We have employed secondary structure prediction, structural superimposition, protein disorderness, molecular docking, and MD simulation to investigate host-pathogen interactions, immune evasion, and transmissibility caused by mutations in the RBD region of the spike protein of the Omicron variant and compared it to the Delta variants (AY.1, AY.2, & AY.3) and wild type. Computational analysis revealed that the Omicron variant has a higher binding affinity for the human ACE2 receptor than the wild and Delta (AY.1 and AY.2 strains), but lower than the Delta AY.3 strain. MD simulation and docking analysis suggest that the omicron and Delta AY.3 were found to have relatively unstable and compact RBD structures and hampered interactions with antibodies more than wild and Delta (AY.1 and AY.2), which may lead to relatively more pathogenicity and antibody escape. In addition, we observed lower binding affinity of Omicron for human monoclonal antibodies (CR3022, B38, CB6, and P2B2F6) when compared to wild and Delta (AY.1 & AY.2). However, the binding affinity of Omicron RBD variants for CR3022, B38, and P2B2F6 antibodies is lower as compared to Delta AY.3, which might promote immune evasion and reinfection and needs further experimental investigation.
Identifying protein sites contributing to vaccine escape via statistical comparisons of short-term molecular dynamics simulations
Authors: Rajendran Madhusudan , Ferran Maureen C. , Babbitt Gregory A.
Published: December 2021

The identification of viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a molecular dynamics (MD) based approach that scales well to a desktop computer with a high-end modern graphics processor and enables the user to identify protein sites that are prone to vaccine escape in a viral antigen. We first implement our MD pipeline to employ site-wise calculation of Kullback-Leibler divergence in atom fluctuation over replicate sets of short-term MD production runs thus enabling a statistical comparison of the rapid motion of influenza hemagglutinin (HA) in both the presence and absence of three well-known neutralizing antibodies. Using this simple comparative method applied to motions of viral proteins, we successfully identified in silico all previously empirically confirmed sites of escape in influenza HA, predetermined via selection experiments and neutralization assays. Upon the validation of our computational approach, we then surveyed potential hot spot residues in the receptor binding domain of the SARS-CoV-2 virus in the presence of COVOX-222 and S2H97 antibodies. We identified many single sites in the antigen-antibody interface that are similarly prone to potential antibody escape and that match many of the known sites of mutations arising in the SARS-CoV-2 variants of concern. In the omicron variant, we find only minimal adaptive evolutionary shifts in the functional binding profiles of both antibodies. In summary, we provide a fast and accurate computational method to monitor hot spots of functional evolution in antibody binding footprints.
Intranasal immunization with a vaccinia virus vaccine vector expressing pre-fusion stabilized SARS-CoV-2 spike fully protected mice against lethal challenge with the heavily mutated mouse-adapted SARS2-N501YMA30 strain of SARS-CoV-2
Authors: Kibler Karen V. , Szczerba Mateusz , Lake Douglas , Roeder Alexa J. , Rahman Masmudur , Hogue Brenda G. , Wong Lok-Yin Roy , Perlman Stanley , Li Yize , Jacobs Bertram L.
Published: December 2021

The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.
SARS-CoV-2 variants of concern remain dependent on IFITM2 for efficient replication in human lung cells
Authors: Nchioua Rayhane , Schundner Annika , Kmiec Dorota , Bozzo Caterina Prelli , Zech Fabian , Koepke Lennart , Graf Alexander , Krebs Stefan , Blum Helmut , Frick Manfred , Sparrer Konstantin M. J. , Kirchhoff Frank
Published: December 2021

The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2021/468942. 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.
The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies
Authors: Chan Conrad En-Zuo , Ng Ching-Ging , Lim Angeline Pei-Chew , Seah Shirley Lay-Kheng , Chye De-Hoe , Wong Steven Ka-Khuen , Lim Jie-Hui , Lim Vanessa Zi-Yun , Lai Soak-Kuan , Wong Pui-San , Leong Kok-Mun , Liu Yi-Chun , Sugrue Richard J , Tan Boon-Huan
Published: December 2021

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection.

Importance

The SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.
Outbreak of COVID-19 among vaccinated and unvaccinated homeless shelter residents — Sonoma County, California, July 2021
Authors: Bukatko Aleksandr , Lobato Mark N. , Mosites Emily , Stainken Cameron , Reihl Katheryn , Deldari Mojgan , Bell John M. , Morris Mary Kate , Wadford Debra A. , Harriman Kathleen , Mase Sundari
Published: December 2021

In July 2021, the Sonoma County Health Department was alerted to three cases of COVID-19 among residents of a homeless shelter in Santa Rosa, California. Among 153 shelter residents, 83 (54%) were fully vaccinated; 71 (86%) vaccinated residents had received the Janssen COVID-19 vaccine and 12 (14%) received an mRNA (Pfizer BioNTech or Moderna) COVID-19 vaccine. Within 1 month, 116 shelter residents (76%) received positive SARS-CoV-2 test results, including 66 fully vaccinated residents and 50 not fully vaccinated. 9 fully vaccinated and 1 unvaccinated were hospitalized for COVID-19. All hospitalized cases had at least one underlying medical condition. Two deaths occurred, one in a vaccinated resident and one in a non-vaccinated resident. Specimens from 52 residents underwent whole genome sequencing; all were identified as SARS-CoV-2, Delta Variant AY.13 lineage. Additional mitigation measures are needed in medically vulnerable congregate setting where limited resources make individual quarantine and isolation not feasible.
Racial and ethnic disparities in COVID-19 disease incidence independent of comorbidities, among people with HIV in the US
Authors: Bender Ignacio RA , Shapiro AE , Nance RM , Whitney BM , Delaney JAC , Bamford L , Wooten D , Karris M , Mathews WC , Kim HN , Van Rompaey SE , Keruly JC , Burkholder G , Napravnik S , Mayer KH , Jacobson J , Saag MS , Moore RD , Eron JJ , Willig AL , Christopoulos KA , Martin J , Hunt PW , Crane HM , Kitahata MM , Cachay E ,   for the Centers for AIDS Research Network of Integrated Clinical Systems (CNICS)
Published: December 2021

Objectives

To define the incidence of clinically-detected COVID-19 in people with HIV (PWH) in the US and evaluate how racial and ethnic disparities, comorbidities, and HIV-related factors contribute to risk of COVID-19.

Design

Observational study within the CFAR Network of Integrated Clinical Systems cohort in 7 cities during 2020.

Methods

We calculated cumulative incidence rates of COVID-19 diagnosis among PWH in routine care by key characteristics including race/ethnicity, current and lowest CD4 count, and geographic area. We evaluated risk factors for COVID-19 among PWH using relative risk regression models adjusted with disease risk scores.

Results

Among 16,056 PWH in care, of whom 44.5% were Black, 12.5% were Hispanic, with a median age of 52 years (IQR 40-59), 18% had a current CD4 count < 350, including 7% < 200; 95.5% were on antiretroviral therapy, and 85.6% were virologically suppressed. Overall in 2020, 649 PWH were diagnosed with COVID-19 for a rate of 4.94 cases per 100 person-years. The cumulative incidence of COVID-19 was 2.4-fold and 1.7-fold higher in Hispanic and Black PWH respectively, than non-Hispanic White PWH. In adjusted analyses, factors associated with COVID-19 included female sex, Hispanic or Black identity, lowest historical CD4 count <350 (proxy for CD4 nadir), current low CD4/CD8 ratio, diabetes, and obesity.

Conclusions

Our results suggest that the presence of structural racial inequities above and beyond medical comorbidities increased the risk of COVID-19 among PWH

PWH with immune exhaustion as evidenced by lowest historical CD4 or current low CD4:CD8 ratio had greater risk of COVID-19.
Current state of the evidence on community treatments for people with complex emotional needs: a scoping review
Authors: Ledden Sarah , Rains Luke Sheridan , Schlief Merle , Barnett Phoebe , Fung Ching Brian Chi , Hallam Brendan , Günak Mia Maria , Steare Thomas , Parker Jennie , Labovitch Sarah , Oram Sian , Pilling Steve , Johnson Sonia ,   CEN Mental Health Policy Research Group
Published: December 2021

Background

Improving the quality of care in community settings for people with ‘Complex Emotional Needs’ (CEN - our preferred working term for services for people with a “personality disorder” diagnosis or comparable needs) is recognised internationally as a priority. Plans to improve care should be rooted as far as possible in evidence. We aimed to take stock of the current state of such evidence, and identify significant gaps through a scoping review of published investigations of outcomes of community-based psychosocial interventions designed for CEN.

Methods

We conducted a scoping review with systematic searches. We searched six bibliographic databases, including forward and backward citation searching, and reference searching of relevant systematic reviews. We included studies using quantitative methods to test for effects on any clinical, social, and functioning outcomes from community-based interventions for people with CEN.

Results

We included 226 papers in all (209 studies). Little relevant literature was published before 2000. Since then, publications per year and sample sizes have gradually increased, but most studies are relatively small, including many pilot or uncontrolled studies. Most studies focus on symptom and self-harm outcomes of various forms of specialist psychotherapy: most result in outcomes better than from inactive controls and similar to other specialist psychotherapies. We found large evidence gaps.

Adaptation and testing of therapies for significant groups (e.g. people with comorbid psychosis, bipolar disorder, post-traumatic stress disorder or substance misuse; older and younger groups; parents) have for the most part only reached a feasibility testing stage. We found little evidence regarding interventions to improve social aspects of people’s lives, peer support or ways of designing effective services.

Conclusions

Compared with other longer term mental health problems that significantly impair functioning, the evidence base on how to provide high quality care for people with CEN is very limited. There is good evidence that people with CEN can be effectively helped when specialist therapies are available and they are able to engage with them. However, a much more methodologically robust and substantial literature addressing a much wider range of research questions is urgently needed to optimise treatment and support across this group.
Proteogenomic analysis of aneuploidy reveals divergent types of gene expression regulation across cellular pathways
Authors: Cheng Pan , Zhao Xin , Katsnelson Lizabeth , Moya Raquel , Shwetar Jasmine , Fenyö David , Davoli Teresa
Published: December 2021

How cells control gene expression is a fundamental question. The relative contribution of protein-level and transcript-level regulation to this process remains unclear. Here we perform a proteogenomic analysis of tumors and untransformed cells containing somatic copy number alterations (SCNAs). By revealing how cells regulate transcript and protein abundances of SCNA-containing genes, we provide insights into the rules of gene regulation. While gene compensation mainly occurs at the protein level across tumor types, genes gained or lost show surprisingly low protein compensation in lung and high RNA compensation in colon cancer. Protein complex genes have a strong protein-level regulation while non-complex genes have a strong transcript-level regulation. Exceptions are plasma membrane protein complexes showing a very low protein-level regulation. Strikingly, we find a strong negative association between the degree of transcript-level and protein-level regulation across genes and pathways. Moreover, genes participating in the same pathway show similar degree of transcript- and protein-level regulation. Pathways including translation, splicing and mitochondrial function show a stronger protein-level regulation while cell adhesion and migration pathways show a stronger transcript-level regulation. These results suggest that the evolution of gene regulation is shaped by functional constraints and that many cellular pathways tend to evolve a predominant mechanism of gene regulation, possibly due to energetic constraints.

Highlights

Proteogenomic analyses of cancer SCNAs reveal tissue specificity in gene compensation.

Genes gained or lost show surprisingly low protein compensation in lung cancer and unexpected RNA compensation in colon cancer.

We use DNA-RNA and RNA-protein correlations to infer the degree of RNA-level and protein-level regulation.

Protein complex genes and non-complex genes show high protein-level and RNA-level regulation, respectively.

Plasma membrane complexes are an exception showing more RNA-level than protein-level regulation than other complex genes.

Genes participating in the same pathway show similar degree of RNA-level and protein-level regulation.

There is a strong negative relationship between the RNA- and protein-level regulation among pathways, suggesting that they are regulated either at the protein or at the RNA level.

Genes involved in RNA processing and protein synthesis are upregulated in highly aneuploid tumors, especially at the protein level.
RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy
Authors: Guichard Marjorie , Holla Sanjana , Wernerová Daša , Grossmann Guido , Minina Elena A.
Published: December 2021

Arabidopsis root is a classic model system in plant cell and molecular biology. The sensitivity of plant roots to local environmental perturbation challenges data reproducibility and incentivizes further optimization of imaging and phenotyping tools.

Here we present RoPod, an easy-to-use toolkit for low-stress live time-lapse imaging of Arabidopsis roots. RoPod comprises a dedicated protocol for plant cultivation and a customizable 3D-printed vessel with integrated microscopy-grade glass that serves simultaneously as a growth and imaging chamber. RoPod reduces impact of sample handling, preserves live samples for prolonged imaging sessions, and facilitates application of treatments during image acquisition.

We describe a protocol for RoPods fabrication and provide illustrative application pipelines for monitoring root hair growth and autophagic activity. Furthermore, we showcase how the use of RoPods advanced our understanding of plant autophagy, a major catabolic pathway and a key player in plant fitness. Specifically, we obtained fine time resolution for autophagy response to commonly used chemical modulators of the pathway and revealed previously overlooked cell type-specific changes in the autophagy response. These results will aid a deeper understanding of the physiological role of autophagy and provide valuable guidelines for choosing sampling time during end-point assays currently employed in plant autophagy research.
Small transcriptional differences among cell clones lead to distinct NF-κB dynamics
Authors: Kizilirmak Cise , Monteleone Emanuele , García-Manteiga José Manuel , Brambilla Francesca , Agresti Alessandra , Bianchi Marco E. , Zambrano Samuel
Published: December 2021

Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at single-cell level, even between very similar cells. We asked how such heterogeneity emerges for the nuclear factor κB (NF-κB), whose dynamics have been reported to cover a wide spectrum of behaviors, including persistent, oscillatory and weak activation. We found that clonal populations of immortalized fibroblasts derived from a single mouse embryo display robustly distinct dynamics upon tumor necrosis factor α (TNF-α) stimulation, which give rise to differences in the transcription of NF-κB targets. Notably, standard transcriptomic analyses indicate that the clones differ mostly in transcriptional programs related with development, but not in TNF-α signaling. However, by combining transcriptomics data and simulations we show how the expression levels of genes coding for proteins of the signaling cascade determine the differences in early NF-κB activation; differences in the expression of IκBα determine differences in its persistence and oscillatory behavior. The same analysis predicts inter-clonal differences in the NF-κB response to IL-1β. We propose that small (less than twofold) differences at transcript level can lead to distinct transcription factor dynamics in cells within homogeneous cell populations, and all the more so among different cell types.
Altricial bird early-stage embryos express the molecular ‘machinery’ to respond to maternal thyroid hormone cues
Authors: Ruuskanen Suvi , Hukkanen Mikaela , Garcin Natacha , Cossin-Sevrin Nina , Hsu Bin-Yan , Stier Antoine
Published: December 2021

Maternal hormones, such as thyroid hormones transferred to embryos and eggs, are key signalling pathways to mediate maternal effects. To be able to respond to maternal cues, embryos must express key molecular ‘machinery’ of the hormone pathways, such as enzymes and receptors. While altricial birds begin thyroid hormone (TH) production only at/after hatching, experimental evidence suggests that their phenotype can be influenced by maternal THs deposited in the egg. However, it is not understood, how and when altricial birds express genes in the TH-pathway. For the first time, we measured the expression of key TH-pathway genes in altricial embryos, using two common altricial ecological model species (pied flycatcher, Ficedula hypoleuca and blue tit Cyanistes caeruleus). Deiodinase DIO1 gene expression could not be reliably confirmed in either species, but deiodinase enzyme DIO2 and DIO3 genes were expressed in both species. Given that DIO2 coverts T4 to biologically active T3, and DIO3 mostly T3 to inactive forms of thyroid hormones, our results suggest that embryos may modulate maternal signals. Thyroid hormone receptor (THRA and THRB) and monocarboxyl membrane transporter gene (SLC15A2) were also expressed, enabling TH-responses. Our results suggest that early altricial embryos may be able to respond and potentially modulate maternal signals conveyed by thyroid hormones.
Large Scale Discovery of Microbial Fibrillar Adhesins and Identification of Novel Members of Adhesive Domain Families
Authors: Monzon Vivian , Bateman Alex
Published: December 2021

Fibrillar adhesins are bacterial cell surface proteins that mediate interactions with the environment including host cells during colonisation or other bacteria during biofilm formation. These proteins are characterised by a stalk that projects the adhesive domain closer to the binding target. Fibrillar adhesins evolve quickly and thus can be difficult to computationally identify, yet they represent an important component for understanding bacterial host interactions.

To detect novel fibrillar adhesins we developed a random forest prediction approach based on common characteristics we identified for this protein class. We applied this approach to Firmicute and Actinobacterial proteomes, yielding over 6,500 confidently predicted fibrillar adhesins. To verify the approach we investigated predicted fibrillar adhesins that lacked a known adhesive domain. Based on these proteins, we identified 24 sequence clusters representing potential novel members of adhesive domain families. We used AlphaFold to verify that 15 clusters showed structural similarity to known adhesive domains such as the TED domain.

Overall our study has made a significant contribution to the number of known fibrillar adhesins and has enabled us to identify novel members of adhesive domain families involved in the bacterial pathogenesis.

Importance

Fibrillar adhesins are a class of bacterial cell surface proteins that enable bacteria to interact with their environment. We developed a Machine Learning approach to identify fibrillar adhesins and applied this classification approach on the Firmicutes and Actinobacteria Reference Proteomes. This method allowed us to detect a high number of novel fibrillar adhesins, and also novel members of adhesive domain families. To confirm our predictions of these potential adhesin protein domains, we predicted their structure using the AlphaFold tool.
HSV-1 and influenza infection induce linear and circular splicing of the long NEAT1 isoform
Authors: Friedl Marie-Sophie , Djakovic Lara , Kluge Michael , Hennig Thomas , Whisnant Adam W. , Backes Simone , Dölken Lars , Friedel Caroline C.
Published: December 2021

The herpes simplex virus 1 (HSV-1) virion host shut-off (vhs) protein cleaves both cellular and viral mRNAs by a translation-initiation-dependent mechanism. Here, we show thatvhs-mediated degradation of mRNAs leads to an accumulation of circular RNAs (circRNAs) relative to linear mRNAs during HSV-1 infection. Strikingly, we found that circular splicing of the long isoform (NEAT1_2) of the nuclear paraspeckle assembly transcript 1 (NEAT1) was massively induced during HSV-1 infection. In contrast to other circRNAs, induction of the NEAT1_2 circRNA was independent ofvhsand occurred while NEAT1_2 was still bound to the chromatin. This was associated with induction of linear splicing of NEAT1_2 both within and downstream of the circRNA. NEAT1_2 splicing was absent in uninfected cells but can be induced by ectopic co-expression of the HSV-1 immediate-early proteins ICP22 and ICP27. Interestingly, NEAT1_2 circular and linear splicing was also up-regulated in influenza A virus (IAV) infection but absent in stress conditions, which disrupt transcription termination similar to but not by the same mechanisms as HSV-1 and IAV infection. Large-scale analysis of published RNA-seq data uncovered induction of NEAT1_2 splicing in cancer cells upon inhibition or knockdown of cyclin-dependent kinase 7 (CDK7) or the MED1 subunit of the Mediator complex phosphorylated by CDK7. Finally, CDK7 inhibition also disrupted transcription termination, highlighting a possible link between disruption of transcription termination and NEAT1_2 splicing.
Structural insights into distinct signaling profiles of the μOR activated by diverse agonists
Authors: Qu Qianhui , Huang Weijiao , Aydin Deniz , Paggi Joseph M. , Seven Alpay B. , Wang Haoqing , Chakraborty Soumen , Che Tao , DiBerto Jeffrey F. , Robertson Michael J. , Inoue Asuka , Roth Bryan L. , Majumdar Susruta , Dror Ron O. , Kobilka Brian K. , Skiniotis Georgios
Published: December 2021

Drugs targeting the G protein-coupled μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression. While some partial opioid agonists appear to be safer than full agonists, the signaling pathways responsible for respiratory depression have yet to be elucidated. Here we investigated the structural and mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most potent and lethal opioids, and MP, a derivative from the kratom plant with reduced respiratory depression in animal studies at equianalgesic doses, exhibited markedly different signaling efficacy profiles for G protein subtype activation and recruitment of β-arrestins. Cryo-EM structures of the μOR-Gi1 complex with MP (2.5Å) and LFT (3.2Å) revealed that the two ligands engage distinct sub-pockets, and molecular dynamics (MD) simulations showed additional differences in the binding site that propagate to the intracellular side of the receptor where G proteins and β-arrestins bind. While MP favors the precise G protein-bound active state observed in the cryo-EM structures, LFT favors a distinct active state. These results highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes.
Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids
Authors: Medina-Cano Daniel , Corrigan Emily K. , Glenn Rachel A. , Islam Mohammed T. , Lin Yuan , Kim Juliet , Cho Hyunwoo , Vierbuchen Thomas
Published: December 2021

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generate target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof-of-concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools within vitrodifferentiation to characterize molecular and cellular mechanisms of embryonic development.

SUMMARY STATEMENT

New optimized protocols for directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain-patterned organoids.
Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis
Authors: Mantri Madhav , Hinchman Meleana M. , McKellar David W. , Wang Michael F. Z. , Cross Shaun T. , Parker John S. L. , De Vlaminck Iwijn
Published: December 2021

A significant fraction of sudden death in children and young adults is due to myocarditis, an inflammatory disease of the heart, most often caused by viral infection. Here we used integrated single-cell and spatial transcriptomics to create a high-resolution, spatially resolved map of reovirus-induced myocarditis in neonatal murine hearts. We assayed hearts collected at three timepoints after reovirus infection and studied the temporal, spatial, and cellular heterogeneity of host-virus interactions. We further assayed the intestine, the primary site of reovirus infection to establish a full chronology of molecular events that ultimately lead to myocarditis. We implemented targeted enrichment of viral transcripts to establish the cellular targets of the virus in the intestine and the heart. Our data give insight into the cell-type specificity of innate immune responses, and into the transcriptional states of inflamed cardiac cells in reovirus-infected heart. We find that inflamed endothelial cells recruit cytotoxic T cells and undergo pyroptosis in the myocarditic tissue. Analyses of spatially restricted gene expression in myocarditic regions and the border zone around those regions identified immune-mediated cell-type specific injury and stress responses. Overall, we observe a dynamic and complex network of cellular phenotypes and cell-cell interactions associated with viral myocarditis.
Neural Oscillatory Characteristics of Feedback Associated Activity in Globus Pallidus Interna
Authors: Choubdar Hadi , Mahdavi Mahdi , Rostami Zahra , Zabeh Erfan , Gillies Martin J. , Green Alexander L. , Aziz Tipu Z. , Lashgari Reza
Published: December 2021

Neural oscillatory activities in basal ganglia have prominent roles in cognitive processes on local and global scales. However, the characteristics of high frequency oscillatory activities during cognitive tasks have not been extensively explored in human Globus Pallidus internus (GPi). This study aimed to investigate amplitude and interhemispheric coupling of bilateral GPi high gamma bursts in dystonia and Parkinson’s Disease (PD) patients, in on and off medication states, after feedback during the Intra-Extra-Dimension shift (IED) task. Bilateral GPi Local Field Potentials (LFP) activity was recorded via externalized DBS electrodes during the IED task. Inter hemisphere phase synchrony was assessed using Inter-Site Phase Clustering (ISPC). Transient high gamma activity (∼100-150Hz) was observed immediately after feedback in the dystonia patient. Moreover, these bursts were phase synchronous between left and right GPis with an antiphase clustering of phase differences. In contrast, no synchronous high gamma activity was detected in the PD patient with or without dopamine administration. The off-med PD patient displayed enhanced low frequency clusters ameliorated by medication in the on-med state. Furthermore, an increased low frequency activity was observed after feedback of incorrect trials in both disease states. The current study provides a rare report of antiphase homotopic synchrony in human GPi, potentially related to incorporating and processing feedback information. The absence of these activities in off and on-med PD indicates the potential presence of impaired medication independent circuits related to feedback processing. Together, these findings are helpful in pointing to the potential role of GPi’s synchronized high frequency activity in cognitive tasks and feedback information processing.
High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations
Authors: Platisa Jelena , Ye Xin , Ahrens Allison M. , Liu Chang , Chen Ichun Anderson , Davison Ian G. , Tian Lei , Pieribone Vincent A. , Chen Jerry L.
Published: December 2021

Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4×0.4mm2 field of view, and; a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise limited signals. Through these combined advances, we achieved simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.
Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations
Authors: Shekhar Mrinal , Gupta Chitrak , Suzuki Kano , Murata Takeshi , Singharoy Abhishek
Published: December 2021

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwith standing the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or ‘dwell’ is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.