bluuebunny/crossref_metadata_2025 huggingface dataset:
Susceptibility to multitasking in chronic stroke is associated to damage of the multiple demand system and leads to lateralized visuospatial deficits
Authors: Blini Elvio , D’Imperio Daniela , Romeo Zaira , De Filippo De Grazia Michele , Passarini Laura , Pilosio Cristina , Meneghello Francesca , Bonato Mario , Zorzi Marco
Published: October 2023

Functional impairment after stroke is related to the amount of brain damage but there is no strict correspondence between lesion and magnitude of the deficit or its recovery. Theoretical constructs such as cognitive or brain reserve have been invoked as unspecific protective factors to explain this mismatch. Here, we consider the opposite point of view, that is the instances in which this protection is lost or overturned. Several studies have shown – in domains encompassing sensory, motor, and cognitive deficits – that paradigms in which the inherent processing limits of the brain are stressed (e.g., by introducing multitasking and attentional load), are indeed capable to unveil the presence of deficits that are otherwise missed. We administered a computerized multitasking paradigm to a sample of 46 consecutive patients with first-ever unilateral subacute to chronic stroke and no sign of lateralized spatial-attentional disorders according to established diagnostic tests. We then used cluster analysis to classify patients, in a purely data-driven manner, according to their multivariate pattern of performance across different conditions (e.g., single- vs dual-tasking, ipsi- vs. contra-lesional stimuli). This enabled us to identify, within a group of putatively spared patients, a cluster of individuals presenting with stark contralesional biases of spatial awareness exclusively in conditions of concurrent attentional load, i.e. a phenotype characterised by high susceptibility to multitasking. This construct was also captured by a latent factor obtained from principal component analysis, providing a continuous susceptibility index across the whole sample. In spite of similar lesion volume, patients in the high susceptibility cluster presented worse performance in activities of daily living and neuropsychological tests evaluating domain-general abilities spanning attention, executive functions, and reasoning. Multivariate predictive modeling based on lesions location and structural disconnections revealed distinctive correlates of high sensitivity to multitasking in the Multiple-Demand (MD) System, a network of frontal and fronto-parietal areas subserving domain general processes. Damage in these areas may critically interact with domain-specific ones (e.g., devoted to spatial attention), resulting in subtle, but significant difficulties for patients in everyday life situations. In conclusion, the construct of susceptibility to multitasking has the promising potential to provide us a better understanding of what marks the passage, after brain damage, to clinically visible deficits.

Graphical Abstract
Linkingin vivomuscle dynamics toin situforce-length and force-velocity reveals that guinea fowl lateral gastrocnemius operates at shorter than optimal lengths
Authors: Schwaner MJ , Mayfield DL , Azizi E , Daley MA
Published: October 2023

Force-length (F-L) and force-velocity (F-V) properties characterize skeletal muscle’s intrinsic properties under controlled conditions, and it is thought that these properties can inform and predictin vivomuscle function. Here, we map dynamicin vivooperating range and mechanical function during walking and running, to the measuredin situF-L and F-V characteristics of guinea fowl (Numida meleagris) lateral gastrocnemius (LG), a primary ankle extensor. We usein vivopatterns of muscle tendon force, fascicle length, and activation to test the hypothesis that muscle fascicles operate at optimal lengths and velocities to maximize force or power production during walking and running. Our findings only partly support our hypothesis:in vivoLG velocities are consistent with optimizing power during work production, and economy of force at higher loads. However, LG does not operate at lengths on the force plateau (±5% Fmax) during force production. LG length was near L0at the time of EMG onset but shortened rapidly such that force development during stance occurred almost entirely on the ascending limb of the F-L curve, at shorter than optimal lengths. These data suggest that muscle fascicles shorten across optimal lengths in late swing, to optimize the potential for rapid force development near the swing-stance transition. This may provide resistance against unexpected perturbations that require rapid force development at foot contact. We also found evidence of passive force rise (in absence of EMG activity) in late swing, at lengths where passive force is zeroin situ, suggesting that dynamic history dependent and viscoelastic effects may contribute toin vivoforce development. Direct comparison ofin vivowork loops and physiological operating ranges to traditional measures of F-L and F-V properties suggests the need for new approaches to characterize dynamic muscle properties in controlled conditions that more closely resemblein vivodynamics.
NODDI-derived measures of microstructural integrity in medial temporal lobe white matter pathways are associated with Alzheimer’s disease pathology and cognitive outcomes
Authors: Parker Dana M. , Adams Jenna N. , Kim Soyun , McMillan Liv , Yassa Michael A. ,   the Alzheimer’s Disease Neuroimaging Initiative
Published: October 2023

INTRODUCTION

Diffusion tensor imaging has been used to assess white matter (WM) changes in the early stages of Alzheimer’s disease (AD). However, the tensor model is necessarily limited by its assumptions. Neurite Orientation Dispersion and Density Imaging (NODDI) can offer insights into microstructural features of WM change. We assessed whether NODDI more sensitively detects AD-related changes in medial temporal lobe WM than traditional tensor metrics.

METHODS

Standard diffusion and NODDI metrics were calculated for medial temporal WM tracts from 199 older adults drawn from ADNI3 who also received PET to measure pathology and neuropsychological testing.

RESULTS

NODDI measures in medial temporal tracts were more strongly correlated to cognitive performance and pathology than standard measures. The combination of NODDI and standard metrics exhibited the strongest prediction of cognitive performance in random forest analyses.

CONCLUSIONS

NODDI metrics offer additional insights into contributions of WM degeneration to cognitive outcomes in the aging brain.
RNF213 variant and autophagic impairment: A pivotal link to endothelial dysfunction in Moyamoya disease
Authors: Shin Hee Sun , Park Geun Hwa , Choi Eun Sil , Park So Young , Kim Da Sol , Chang Jaerak , Hong Ji Man
Published: October 2023

Background

Moyamoya disease (MMD) is closely associated with the Ring Finger Protein 213 (RNF213), a susceptibility gene for this disease. However, its biological function remains unclear. We aimed to elucidate the role of RNF213 in the damage incurred by human endothelial cells under oxygen-glucose deprivation (OGD), a condition that mimics intracranial ischemia in patients with MMD.

Methods

We analyzed autophagy in peripheral blood mononuclear cells (PBMCs) derived from patients carrying either RNF213 wild-type (WT) or variant (R4810K). Subsequently, human umbilical vein endothelial cells (HUVECs) were transfected with RNF213 WT (HUVECWT) or R4810K (HUVECR4810K) and exposed to OGD for 2 h to determine the role of the RNF213 variant in such a setting. Immunoblotting was used to analyze autophagy marker proteins, and tube formation assays were performed to examine endothelial function. Autophagic vesicles were observed using transmission electron microscopy. Post-OGD exposure, we administered autophagy modulators such as rapamycin and cilostazol.

Results

The RNF213 variant group during post-OGD exposure (vs. pre-OGD exposure) showed autophagy inhibition, increased protein expression of SQSTM1/p62 (p< 0.0001) and LC3-II (p= 0.0039), and impaired endothelial function (p= 0.0252). HUVECR4810Kduring post-OGD exposure (versus pre-OGD exposure) showed a remarkable increase in autophagic vesicles. Administration of autophagy modulators notably restored the function of HUVECR4810Kand cellular autophagy.

Conclusions

Our findings support the pivotal role of autophagy impaired by the RNF213 variant in MMD-induced endothelial cell dysfunction and underscore the critical mechanism of autophagy leading to progressive endothelial dysfunction and MMD pathogenesis under relative ischemia within the intracranial portion.
Metabolomics reveals reasons for the efficacy of acupuncture in migraine patients: The role of anaerobic glycolysis and mitochondrial citrate in migraine relief
Authors: Gao Zishan , Yan Xianzhong , Wang-Sattler Rui , Covic Marcela , Yu Guang , Ge Feifei , Lin Jia , Chen Qin , Liu Juan , Sharma Sapna , Molnos Sophie , Kuehnel Brigitte , Wilson Rory , Adam Jonathan , Brandmaier Stefan , Yu Shuguang , Mansmann Ulrich , Liang Fanrong , Gieger Christian
Published: October 2023

Acupuncture is used worldwide to treat migraine, but its scientific mechanism remains unclear. Here, we report a 1H NMR metabolomics study involving 40 migraine patients and 10 healthy people randomly receiving acupuncture or sham acupuncture, followed by machine learning techniques and functional analysis. We found that acupuncture at acupoints particularly enhanced anaerobic glycolysis and modified mitochondrial function by adjusting the levels of plasma pyruvic acid (P = 0.012), lactic acid (P = 0.031) and citrate (P = 0.00079) at a Bonferroni-corrected level of significance compared to the pre-treatment level of these three metabolites in migraine patients. Therefore, acupuncture supplies energy to migraine patients and relieves migraine attacks. In contrast, we observed that sham acupuncture may partially supply energy to migraine patients through lipid metabolism by changing the levels of plasma lipid (P = 0.0012), glycerine (P = 0.021), and pyruvic acid (P = 0.047) at a Bonferroni-corrected level of significance. The functional network analysis further indicates this different way of supplying energy contributes to the different effects of acupuncture and sham acupuncture. Our findings reveal novel metabolic evidence for the specific effect of acupuncture in relation to sham acupuncture. This metabolic evidence could enlighten a brand new direction into acupuncture analgesia mechanism, which in turn would pose fresh challenges for future acupuncture research.
The Psychological Impact of Torture and State Repression in Türkiye between 2015 and 2018: Reports from Turkish Refugees Seeking Asylum in Germany
Authors: Tambini Stollwerck Estella Alejandra , Sarikaya Ilkem , Yen Kathrin , Friederich Hans-Christoph , Nikendei Christoph
Published: October 2023

Torture seeks to undermine not only the physical and emotional well-being of an individual but to damage the coherence of entire communities. Thus, torture and state repression are used to weaken entire subpopulations. After the failed coup d’état in Türkiye in 2016 and during the subsequently following state of emergency that lasted until 2018, allegations of torture and other degrading treatment in Türkiye spread widely. Since then, the number of asylum-seekers in Germany has risen considerably.

This paper analyses the reports of twenty Turkish citizens that fled to Germany to seek asylum in the aftermath of the events. In semi-structured interviews held in Turkish, we assessed the experiences of torture and state repression, psychological consequences, and the current well-being and living situation.

All interviewees described illicit experiences with violence from state authorities and government supporters, especially while under arrest. Though the methods varied, there was a constant pattern of imbalance of power. The psychological impact of these methods were present after relocation in Germany and included signs of PTSD, anxiety disorders, and major depression.

The reports of torture, state repression, and their psychological impact emphasise the importance for policy makers to address the prevention of human rights violations and support the needs of survivors.
Prevalence of Depression among Senior Secondary School Students in Enugu Metropolis
Authors: Okeke Nzubechukwu Gift , Okenyi Johncross Ogbonna , Ohanu Eberechi Cyprian , Okeke Chidera
Published: October 2023

Background

Depression is defined as a state of low mood and aversion to activity. A mental health disorder characterized by persistent low mood or loss of interest in activities causing significant impairment in daily life. Depression amongst secondary school students is an issue that is often ignored. It is frequently unrecognized when present in adolescents who make up the population of secondary school students (age 10-19 years). In our environment, studies have reported that 21.2% of Nigerian adolescents are suffering from mild to moderate depression and depression when left unchecked and untreated, takes a toll on the quality of life of individuals affected. This is why this study was conducted to determine the prevalence of depression among senior secondary school students in Enugu metropolis, to create proper awareness and sensitize people involved and the public on this menace.

Method

A descriptive cross-sectional study was carried out with a sample size of 270 secondary school students across 10 secondary schools in Enugu metropolis. The population of 270 students was sampled using simple random sampling. All met the inclusion criteria. A self-administered semi-structured questionnaire was used for data collection. Data was analyzed using statistical package for social sciences (SPSS) version 22. Data was presented using tables.

Result

The study had a 100% response rate. Results showed the prevalence of mild depression amongst the respondents as 58.1%, the prevalence of moderate depression amongst the respondents as 31.1% while the prevalence of severe depression was 10.7%. The study found the prevalence of depression to be higher in older adolescents and females. There was also a significant relationship between satisfaction with academic performance and depression.

Conclusion

There was significant level of prevalence of depression among senior secondary school students in Enugu metropolis, notably among the females, older adolescents and those who were not satisfied with their academic performance.

Recommendations

Depression should be seen as what it is, a disease and awareness should be carried out to adequately sensitize the involved population on early recognition of signs and symptoms of depression, appropriate support and therapy and subsequent rehabilitation of depressed individuals.
A lightweight data-driven spiking neural network model ofDrosophilaolfactory nervous system with dedicated hardware support
Authors: Nanami Takuya , Yamada Daichi , Someya Makoto , Hige Toshihide , Kazama Hokto , Kohno Takashi
Published: October 2023

Data-driven spiking neural network (SNN) models are vital for understanding the brain’s information processing at the cellular and synaptic level. While extensive research has focused on developing data-driven SNN models for mammalian brains, their complexity poses challenges in achieving precision. Network topology often relies on statistical inference, and the functions of specific brain regions and supporting neuronal activities remain unclear. Additionally, these models demand significant computational resources. Here, we propose a lightweight data-driven SNN model that strikes a balance between simplicity and reproducibility. We target theDrosophilaolfactory nervous system, extracting its network topology from connectome data. The model implemented on an entry-level field-programmable gate array successfully reproduced the functions and characteristic spiking activities of different neuron types. Our approach thus provides a foundation for constructing lightweightin silicomodels that are critical for investigating the brain’s information processing mechanisms at the cellular and synaptic level through an analysis-by-construction approach and applicable to edge artificial intelligence (AI) systems.
Nanobodies against SARS-CoV-2 non-structural protein Nsp9 inhibit viral replication by targeting innate immunity
Authors: Venit Tomas , Blavier Jeremy , Maseko Sibusiso B. , Shu Sam , Espada Lilia , Breunig Christopher , Holthoff Hans-Peter , Desbordes Sabrina C. , Lohse Martin , Esposito Gennaro , Twizere Jean-Claude , Percipalle Piergiorgio
Published: October 2023

Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not target it to their full potential and as a consequence, inhibition of viral entry may not be efficient. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for viral genome replication. To find out if this strategy efficiently blocks viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA- 2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells with multiple SARS-CoV-2 variants and subjected them to LNP-mRNA-2NSP23 treatment. Analysis of total RNA isolated from infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.
Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin
Authors: Oevel Kristine , Hohensee Svea , Kumar Atul , Rosas-Brugada Irving , Bartolini Francesca , Soykan Tolga , Haucke Volker
Published: October 2023

Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin.
The RNA binding protein, HNRNPA2B1, regulates IFNG signaling in macrophages
Authors: Salih Mays Mohammed , Weindel Chi G , Malekos Eric , Sudek Lisa , Katzman Sol , Mabry Cory J , Coleman Aja K , Azam Sikandar , Watson Robert , Patrick Kristin , Carpenter Susan
Published: October 2023

Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) is a well known RNA binding protein but the mechanisms by which it contributes to innate immune gene regulation are poorly understood. Here we report that HNRNPA2B1 functions in macrophages to regulate IFNG (IFN-γ) signaling through alternative splicing of the IFNG receptor. Specific deletion of HNRNPA2B1 in macrophages resulted in altered cytokine responses in both an endotoxic shock model and followingSalmonellainfection. Interestingly, while HNRNPA2B1 can function as a viability gene, we observed increased macrophage and neutrophil numbers in the KO mice following LPS induced endotoxic shock. We also discovered that HNRNPA2B1 restricts replication ofSalmonella enterica in vivo. Mechanistically, loss of HNRNPA2B1 resulted in an increase in NGO transcripts, which lack a start codon, of the IFNG receptor (Ifngr) leading to lower expression of the receptor at the cell surface impacting the downstream IFNG signaling cascade. Collectively, our data highlight an important role for HNRNPA2B1 in regulating IFNG signaling and restricting intracellular bacterial pathogens in macrophages.
Multiparameter flow cytometric detection and analysis of rare cellsin in vivomodels of cancer metastasis
Authors: Mallin Mikaela M , Pienta Kenneth J. , Amend Sarah R.
Published: October 2023

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection forms a major underpinning of rigorousin vivometastasis research. While many cancer cells initiate metastasis, very few can complete it. Clinical data evidences that each successive step of metastatic cascade presents increasing barriers to metastatic success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical to employ metastasis research approaches that allow scientists to discern which step(s) of the cascade present metastatic barriers to their model systems, and in which steps their model systems might display competency. Here, we present a novel flow-cytometry based method that allows for the simultaneous comparison of multiple steps of the cascade within one model system via the co-identification of CTC and DTCs from single animals. This approach is not only highly reliable and reproducible, but also broadly applicable and highly adaptable to a wide range of scientific inquiries.
Activation of automethylated PRC2 by dimerization on chromatin
Authors: Sauer Paul V. , Pavlenko Egor , Cookis Trinity , Zirden Linda C. , Renn Juliane , Singhal Ankush , Hunold Pascal , Hoehne Michaela N. , van Ray Olivia , Hänsel-Hertsch Robert , Sanbonmatsu Karissa Y. , Nogales Eva , Poepsel Simon
Published: October 2023

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit EZH2 stimulates its activity by an unknown mechanism. Here, we show that PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosterictrans-autoactivation via EED, suggesting a novel mechanism mediating context- dependent activation of PRC2. Our work showcases the molecular mechanism of auto- modification coupled dimerization in the regulation of chromatin modifying complexes.
Prevalence of Autoimmune Diseases in Functional Neurological Disorder: Influence of Psychiatric Comorbidities and Biological Sex
Authors: Joseph Anna , Baslet Gaston , Neal Mary A O’ , Polich Ginger R , Gonsalvez Irene , Christoforou Andrea N , Dworetzky Barbara A , Spagnolo Primavera A
Published: October 2023

Background

Functional neurological disorder (FND) is a common and disabling neuropsychiatric condition, which disproportionally affects women compared to men. While the etiopathogenesis of this disorder remains elusive, immune dysregulation is emerging as one potential mechanism.

Aim

To begin to understand the role of immune dysfunction in FND, we assessed the prevalence of several common autoimmune diseases (ADs) in a large cohort of patients with FND and examined the influence of psychiatric comorbidities and biological sex.

Methods

Using a large biorepository database (Mass General Brigham Biobank), we obtained demographic and clinical data of a cohort of 643 patients diagnosed with FND between January 2015 and December 2021. The proportion of ADs was calculated overall, by sex and by presence of psychiatric comorbidities.

Results

The overall prevalence of ADs in our sample was 41.9%, with connective tissue and autoimmune endocrine diseases being the most commonly observed ADs. Among patients with FND and ADs, 27.7% had ≥2 ADs and 8% met criteria for multiple autoimmune syndrome. Rates of ADs were significantly higher in subjects with comorbid major depressive disorder and post-traumatic stress disorder (p=□0.02). Women represented the largest proportion of patients with concurrent ADs, both in the overall sample and in the subgroups of interest (p’s□<□0.05).

Conclusions

This study is unique in providing evidence of an association between FND and ADs. Future studies are needed to investigate the mechanisms underlying this association and to understand whether FND is characterized by distinct dysregulations in immune response.
Diversity, Equity, and Inclusion or Bigotry, Inequity, and Exclusion? Applicant Perceptions of Obstetrics and Gynecology Residency Programs
Authors: Hale Corinne M. , Cowley Elise S. , Hebgen Shanaya , Spencer Ryan , Jacques Laura
Published: October 2023

Introduction

Residency applicants often evidence of Obstetrics and Gynecology (ObGyn) residency programs’ commitments to diversity, equity, and inclusion. Online peer-to-peer discussions suggest applicants evaluate program commitment to diversity, equity, and inclusion through formal and informal channels and share their insights and experiences to help each other form opinions about program culture.

Methods

The study team examined applicant descriptions of ObGyn residency programs using qualitative data collected from a public resource available on the social media platform, Reddit. Using an inductive approach, the team analyzed anonymous comments in a shared document called “2020-2021 OB GYN Residency Applicant Spreadsheet” housed within Reddit to better understand applicants’ experiences with and perceptions of US ObGyn residency programs. Codes were collectively determined and assigned to 731 unique comments; this paper focuses on the codes directly and indirectly related to diversity, equity, and inclusion as well as racism, discrimination, and bigotry.

Results

We found that applicants used visual, verbal, and behavioral cues to analyze ObGyn residency programs. Students primarily used perceptions gained in program interview days, social events, faculty social media profiles, and program websites to decipher programs’ values and internal commitment to pursuing diversity, equity, and inclusion.

Conclusions

This study emphasizes the value applicants place on residency programs’ commitments to DEI. Programs that can clearly demonstrate their dedication to DEI principles are more likely to attract a diverse applicant pool that will become the future medical workforce. Programs can provide informative context related to DEI undertakings, reducing the risk that applicants will draw erroneous conclusions from partial information.
Genetically refactoredAgrobacterium-mediated transformation
Authors: Thompson Mitchell G. , Kirkpatrick Liam D. , Geiselman Gina M. , Waldburger Lucas M. , Pearson Allison N. , Szarzanowicz Matthew , Vuu Khanh M. , Markel Kasey , Hummel Niklas F. C. , Suazo Dennis D. , Tahmin Claudine , Cui Ruoming , Liu Shuying , Cevallos Jasmine , Pannu Hamreet , Liu Di , Gin Jennifer W. , Chen Yan , Petzold Christopher J. , Gladden John M. , Keasling Jay D. , Chang Jeff H. , Weisberg Alexandra J. , Shih Patrick M.
Published: October 2023

Members ofAgrobacteriumare costly plant pathogens while also essential tools for plant transformation. ThoughAgrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and its complex transcriptional regulation make identifying the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here we use a bottom-up synthetic approach to systematically refactor the tumor-inducing plasmid, wherein the majority of AMT machine components are encoded, into a minimal set of genes capable of plant and fungal transformation that is both controllable and orthogonal to its environment. We demonstrate that engineered vectors can be transferred to new heterologous bacteria, enabling them to transform plants. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, and may lead to the development of strains of bacteria more capable of transforming recalcitrant plant species of societal importance.
Human pluripotent stem cell-derived hepatocyte-like cells for hepatitis D virus studies
Authors: Chi Huanting , Qu Bingqian , Prawira Angga , Maurer Lars , Hu Jungen , Fu Rebecca M. , Lempp Florian A. , Zhang Zhenfeng , Grimm Dirk , Wu Xianfang , Urban Stephan , Dao Thi Viet Loan
Published: October 2023

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co- infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively secrete infectious progeny virions. We also show that HLCs expressing HBsAg support extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV/HBV co-entry factor, which was rate-limiting HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

Teaser

A human stem cell-derived hepatocyte culture model for hepatitis D virus studies
Enhancing a multipurpose artificial urine for culture and gene expression studies of uropathogenicEscherichia colistrains
Authors: Rimbi Patricia T. , O’Boyle Nicky , Douce Gillian R. , Pizza Mariagrazia , Rosini Roberto , Roe Andrew J.
Published: October 2023

UropathogenicEscherichia coli(UPEC) are the most common cause of urinary tract infections, which pose a great burden on global health and the economy through morbidity, mortality, healthcare costs and loss of productivity. Pooled human urine can be used as a growth medium forin vitrostudies, however even if the same donors are used, composition can vary depending largely on diet and fluid intake. There have been a number of artificial urine formulae used as alternatives to pooled human urine. However, we observed that a recently reported multipurpose artificial urine was unable to support the growth of prototypic UPEC strains suggesting it lacked key metabolites. We therefore used liquid chromatography mass spectrometry to identify and adjust the metabolic profile of multipurpose artificial urine closer to that of pooled human urine. Modification in this way facilitated growth of UPEC strains with growth rates similar to those obtained in pooled human urine. Transcriptomic analysis of UPEC strains cultured in enhanced artificial urine and pooled human urine showed that the gene expression profiles are similar, with less than 7% of genes differentially expressed between the two conditions. The data support this enhanced artificial urine as a robust media to study aspects of UPEC physiologyin vitro.
Fucose is an essential feature in cryoprotective polysaccharides
Authors: Guerreiro B. M. , Concórdio-Reis P. , Pericão H. , Martins F. , Moppert X. , Guézennec J. , Lima J.C. , Silva J.C. , Freitas F.
Published: October 2023

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Our current investigation studied 26 natural polysaccharides as potential CPA, probing correlations between post-thaw metabolic viability (PTV) of cryopreserved Vero cells and monomeric composition. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18–35.7 mol%), uronic acids (UA) (13.5–26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. To deconvolute multiple variable effects, principal component analysis (PCA) coupled toK-means clustering was performed. Two major mechanisms of action explained PTV variability: a charge-dependent effect of contrasting charged uronic acid and neutral monomer compositions, and a MW-scaled charge-independent mechanism exclusively attributed to fucose. Ultimately, our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection, highlighting the potential of fucose-rich polyanionic polysaccharides.
Small RNA regulation of an essential process induces bacterial resistance to aminoglycosides during oxidative stress
Authors: Baussier Corentin , Oriol Charlotte , Durand Sylvain , Py Beatrice , Mandin Pierre
Published: October 2023

Fe-S clusters are essential cofactors involved in many reactions across all domains of life. InEscherichia coliand other enterobacteria, Fe-S cluster synthesis involves two machineries: Isc and Suf. While Isc functions as a housekeeping system, Suf is activated under stress conditions such as iron starvation or oxidative stress. Interestingly, cells functioning under Suf show reduced entry of aminoglycosides, leading to resistance to these antibiotics. The transcriptional regulator IscR, itself an Fe-S cluster containing protein, controls the transition between Isc and Suf machineries. Noteworthy, IscR has a critical impact on the virulence of various bacterial pathogens by regulating both Fe-S biogenesis and other pathways directly linked to host adaptation. Here, we discovered that two small regulatory RNAs (sRNAs), FnrS and OxyS, controliscRexpression by base-pairing to the 5’ UTR of theiscRmRNA. Remarkably, these sRNAs act in opposite ways and in opposite conditions: FnrS, expressed in anaerobiosis, represses the expression ofiscRwhile OxyS, expressed during oxidative stress, activates it. Using anE. colistrain experiencing protracted oxidative stress, we further demonstrate thatiscRexpression is rapidly and significantly enhanced in the presence of OxyS. Strikingly, we further show that OxyS induces resistance to aminoglycosides during oxidative stress through this unexpected regulation of Fe-S clusters biogenesis, revealing a new role for this sRNA.

Significance Statement

This study sheds light on the regulatory mechanisms controlling the synthesis of essential Fe-S clusters in bacteria, revealing unexpected roles for two small RNAs (FnrS and OxyS) in modulating the expression of the transcriptional regulator IscR. The findings suggest that this regulatory network could lead bacterial resistance to aminoglycoside antibiotics during oxidative stress, a condition associated with chronic infections. Ultimately, this work highlights the importance of understanding the intricate regulatory networks controlling bacterial metabolism and adaptation to stress, which could have significant implications for public health.
FibrilPaint to determine the length of Tau amyloids in fluids
Authors: Aragonès Pedrola Júlia , Dekker Françoise A. , Garfagnini Tommaso , Mayer Guy , Koopman Margreet B. , Bergmeijer Menno , Förster Friedrich , Hoozemans Jeroen J. M. , Jensen Henrik , Friedler Assaf , Rüdiger Stefan G. D.
Published: October 2023

Tau aggregation into amyloid fibrils is linked to the development of neurodegenerative diseases, including Alzheimer’s Disease. The molecular processes underlying aggregation in disease are poorly understood. Here, we introduce FibrilPaint1 as a tool to measure the size of Tau amyloid fibrils in fluids, from early aggregation stages to mature fibrils. FibrilPaint1 is a 22mer peptide with many exciting properties, which makes it a tool for diagnostics and an attractive start point for developing a class of effective fibril targeting degraders: (i) FibrilPaint1 binds fibrils with nanomolar affinity; (ii) it does also bind to oligomeric precursors, down to a size of only 4 layers; (iii) it does not bind to monomers (KD > 100 µM); (iv) it is fluorescently labelled, which allows monitoring and localising interactions. (v) FibrilPaint1 recognises various Tau fibrils, including patient derived fibrils from Alzheimer, Corticobasal degeneration and Frontotemporal dementia; (vi) FibrilPaint1 is selective for the amyloid state and does not have background binding to amorphous aggregates, blood serum or cell lysate. In combination with Flow Induced Dispersion Analysis (FIDA), a microfluidics technology, we determined the molecular size of amyloid fibrils with sub-microliter sample volumes. This set-up acts as a molecular ruler at layer resolution - we determined Tau fibril length from 4 to 1100 layers in solution. This is an interesting parameter that can be used for diagnostic applications and biochemical research in dementia.
RBM39 shapes innate immunity through transcriptional and splicing control of key factors of the interferon response
Authors: Li Teng-Feng , Rothhaar Paul , Lang Arthur , Grünvogel Oliver , Colasanti Ombretta , Olivera Ugarte Santa Mariela , Traut Jannik , Piras Antonio , Acosta-Rivero Nelson , Gonçalves Magalhães Vladimir , Springer Emely , Betz Andreas , Huang Hao-En , Park Jeongbin , Qiu Ruiyue , Gnouamozi Gnimah Eva , Mehnert Ann-Kathrin , Dao Thi Viet Loan , Urban Stephan , Muckenthaler Martina , Schlesner Matthias , Wohlleber Dirk , Binder Marco , Bartenschlager Ralf , Pichlmair Andreas , Lohmann Volker
Published: October 2023

RNA-binding motif protein 39 (RBM39) is an RNA-binding protein involved in tumorigenesis, cell metabolism, and development. Here, we performed a genome-wide CRISPR/Cas9 screen in two liver-derived cell lines and identified RBM39 as a regulator of cell intrinsic innate immune responses. The knockdown ofRBM39or the treatment with Indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections upon sensing by toll-like receptor 3 (TLR3) or cytosolic RIG-I-like receptors. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion.RBM39knockdown further restrained type I and type III IFN pathways, by reducing expression of the type I IFN receptor subunit interferon alpha and beta receptor subunit 2 (IFNAR2), type III IFN receptor subunit interleukin 10 receptor subunit beta (IL-10RB) and transcription factor signal transducer and activator of transcription (STAT) 1 and 2. RBM39 overall orchestrates innate immunity by regulating basal expression of key factors of the interferon response via transcription and/or alternative splicing.

Significance

The function of RBM39 in tumorigenesis has been investigated intensively in the last decade, but its immunological role is still largely unknown. In our study, we identified RBM39 as a regulatory factor of cell intrinsic signaling via a CRISPR/Cas9 screen. Depletion of RBM39 impairs TLR3, RIG-I/MDA5, and IFN pathways, and thus attenuates innate immune responses. Our omics analysis revealed that RBM39 governs the basal expression of several key factors within these pathways, such as RNA sensors RIG-I and MDA5, type I/III receptors, transcription factors IRF3, STAT1 and STAT2, via its transcriptional and splicing function. Therefore, RBM39 might be a therapeutic target to modulate innate immunity, e.g. in the context of autoimmune disorders.
Disrupting the interaction between AMBRA1 and DLC1 is a promising therapeutic strategy for neurodegeneration that prevents apoptosis while enhancing autophagy and mitophagy
Authors: Hawkins Kate , Watt Meg , Gillotin Sébastien , Hanspal Maya , Helley Martin , Richardson Jill , Corbett Nicola , Brownlees Janet
Published: October 2023

Activating molecule in Beclin1-regulated autophagy (AMBRA1) has critical roles in autophagy, mitophagy, cell cycle regulation, neurogenesis and apoptosis. Dysregulation of these processes are hallmarks of various neurodegenerative diseases and therefore AMBRA1 represents a potential therapeutic target. The flexibility of its intrinsically disordered regions allows AMBRA1 to undergo conformational changes and thus perform its function as an adaptor protein for various different complexes. Understanding the relevance of these multiple protein-protein interactions will allow us to gain information about which to target pharmacologically. To compare potential AMBRA1 activation strategies we have designed and validated several mutant constructs (ACTA, TAT, WD40 and S1014) in addition to characterising their effects on proliferation, apoptosis, autophagy and mitophagy in SHSY5Y cells. AMBRA1TAT, which is a mutant form of AMBRA1 that can’t interact with dynein light chain (DLC)1 at the microtubules, produced the most promising results. Indeed, overexpression of this mutant protected cells against apoptosis and induced autophagy and mitophagy in SHSY5Y cells in addition to enhancing the switch from quiescence to proliferation in mouse neural stem cells. Future studies should focus on designing compounds that inhibit the protein-protein interaction between AMBRA1 and DLC1 and thus have potential to be used as a drug strategy to treat neurodegeneration.
Explainable protein function annotation using local structure embeddings
Authors: Derry Alexander , Altman Russ B.
Published: October 2023

The rapid expansion of protein sequence and structure databases has resulted in a significant number of proteins with ambiguous or unknown function. While advances in machine learning techniques hold great potential to fill this annotation gap, current methods for function prediction are unable to associate global function reliably to the specific residues responsible for that function. We address this issue by introducing PARSE (Protein Annotation by Residue-Specific Enrichment), a knowledge-based method which combines pre-trained embeddings of local structural environments with traditional statistical techniques to identify enriched functions with residue-level explainability. For the task of predicting the catalytic function of enzymes, PARSE achieves comparable or superior global performance to state-of-the-art machine learning methods (F1 score > 85%) while simultaneously annotating the specific residues involved in each function with much greater precision. Since it does not require supervised training, our method can make one-shot predictions for very rare functions and is not limited to a particular type of functional label (e.g. Enzyme Commission numbers or Gene Ontology codes). Finally, we leverage the AlphaFold Structure Database to perform functional annotation at a proteome scale. By applying PARSE to the dark proteome—predicted structures which cannot be classified into known structural families—we predict several novel bacterial metalloproteases. Each of these proteins shares a strongly conserved catalytic site despite highly divergent sequences and global folds, illustrating the value of local structure representations for new function discovery.
Facilitating clinically relevant skin tumor diagnostics with spectroscopy-driven machine learning
Authors: Andersson Emil , Hult Jenny , Troein Carl , Stridh Magne , Sjögren Benjamin , Pekar-Lukacs Agnes , Hernandez-Palacios Julio , Edén Patrik , Persson Bertil , Olariu Victor , Malmsjö Malin , Merdasa Aboma
Published: October 2023

In the dawning era of artificial intelligence (AI), health care stands to undergo a significant transformation with the increasing digitalization of patient data. Digital imaging, in particular, will serve as an important platform for AI to be implemented to aid decision making and diagnostics. A growing number of studies demonstrate the potential of AI for automatic pre-surgical skin tumor delineation, which could have tremendous impact on clinical practice. However, current methods have the drawback of relying on a ground truth image in which the tumor borders are already identified, which is not clinically possible. We report a novel approach where hyperspectral images provides spectra from small regions representing healthy tissue and tumor, which are used to generate prediction maps using artificial neural networks. Thereafter, a segmentation algorithm automatically manages to determine the skin tumor borders. Our approach therefore circumvents the need for a complete ground truth image, where the training data is contained within each individual patient. This links to an important strength of our approach as we develop individual network models for each patient. Our approach is therefore not only more clinically relevant, but it also interesting for emerging precision skin tumor diagnostics where adaptability toward the individual is key.
Influence of myosin regulatory light chain and myosin light chain kinase on hair cells of the inner ear
Authors: Oya Ryohei , Woo Kwang Min , Fabella Brian , Alonso R. G. , Hudspeth A. J.
Published: October 2023

In the receptor organs of the inner ear and lateral line, sensory hair cells detect mechanical stimuli such as sounds, accelerations, and water movements. In each instance a stimulus deflects the hair bundle, a hair cell’s mechanically sensitive organelle. The bundle pivots upon the cell’s apical surface, which includes an actin meshwork called the cuticular plate and is surrounded by a ring of filamentous actin and non-muscle myosin II (NM2). Myosin regulatory light chain (RLC) is expressed at the apical surfaces of hair cells and RLC is additionally found in hair bundles. NM2 and the phosphorylation of RLC by myosin light chain kinase (MLCK) have earlier been shown to regulate the sizes and shapes of hair cells’ apical surfaces. We have found that inhibitors of NM2 and MLCK reduce the stiffness of hair bundles from the bullfrog’s sacculus. Moreover, MLCK inhibition inhibits the spontaneous oscillation of hair bundles and increases the resting open probability of transduction channels. In addition, MLCK inhibition elevates hearing thresholds in mice. We conclude that NM2 and the phosphorylation of RLC modulate slow adaptation and thereby help to set the normal operating conditions of hair bundles.

Statement of significance

Sensory hair cells play a key role in mechanoelectrical transduction by the inner ear and lateral-line system. To detect stimuli such as sounds and accelerations, hair cells use an active process to amplify their mechanical inputs. Although amplification is accomplished in part by the activity of the mechanosensitive hair bundles, the molecular mechanism of the active process remains uncertain. The present study shows that non-muscle myosin II (NM2) and the phosphorylation of myosin regulatory right chains (RLC) by myosin light chain kinase (MLCK) regulate the stiffness and spontaneous oscillation of hair bundles as well as the resting open probability of mechanotransduction channels. The results implicate myosin motors in the control of the active process of hair cells.