bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple conformational states assembly of multidomain proteins using evolutionary algorithm based on structural analogues and sequential homologues
Authors: Peng Chunxiang , Zhou Xiaogen , Liu Jun , Hou Minghua , Li Stan Z. , Zhang Guijun
Published: January 2023

With the breakthrough of AlphaFold2, nearly all single-domain protein structures can be built at experimental resolution. However, accurate modelling of full-chain structures of multidomain proteins, particularly all relevant conformations for those with multiple states remain challenging. In this study, we develop a multidomain protein assembly method, M-SADA, for assembling multiple conformational states. In M-SADA, a multiple population-based evolutionary algorithm is proposed to sample multiple conformational states under the guidance of multiple energy functions constructed by combining homologous and analogous templates with inter-domain distances predicted by deep learning. On a developed benchmark dataset containing 72 multidomain proteins with multiple conformational states, the performance of M-SADA is significantly better than that of AlphaFold2 on multiple conformational states modelling, where 29/72 (40.3%) of proteins can be assembled with a TM-score >0.90 for highly distinct conformational states with M-SADA while AlphaFold2 does so in only 2/72 (2.8%) of proteins. Furthermore, M-SADA is tested on a developed benchmark dataset containing 296 multidomain proteins with single conformational state, and results show that the average TM-score of M-SADA on the best models is 0.913, which is 5.2% higher than that of AlphaFold2 models (0.868).
Pilot Analysis of Genetic Effects on Personality Test Scores with AI: ABO Blood Type in Japan
Authors: Kanazawa Masayuki
Published: January 2023

It is estimated that genetic factors are responsible for approximately 30-60% of an individual’s personality. However, statistical analyses have yet to demonstrate a significant and consistent relationship between personality tests and genetic factors. Conversely, a significant portion of individuals in Japan, South Korea, and Taiwan believe that there is a linkage between ABO blood type, which is determined genetically, and personality. This pilot study analyzed data from a large-scale survey (N=2,887) using a combination of traditional statistical methods and AI to examine this relationship. The results indicated the relationship between ABO blood type and self-reported personality traits on several single-question items, in accordance with expected outcomes. These findings suggest that this relationship may extend beyond ABO blood type and personality to encompass other inherited characteristics. The influence of the level of interest on personality and was also discussed.
Dissecting the Functional Organization of theC. elegansSerotonergic System at Whole-Brain Scale
Authors: Dag Ugur , Nwabudike Ijeoma , Kang Di , Gomes Matthew A. , Kim Jungsoo , Atanas Adam A. , Bueno Eric , Estrem Cassi , Pugliese Sarah , Wang Ziyu , Towlson Emma , Flavell Steven W.
Published: January 2023

Serotonin controls many aspects of animal behavior and cognition. But how serotonin acts on its diverse receptor types in neurons across the brain to modulate global activity and behavior is unknown. Here, we examine how serotonin release from a feeding-responsive neuron inC. elegansalters brain-wide activity to induce foraging behaviors, like slow locomotion and increased feeding. A comprehensive genetic analysis identifies three core serotonin receptors that collectively induce slow locomotion upon serotonin release and three others that interact with them to further modulate this behavior. The core receptors have different functional roles: some induce behavioral responses to sudden increases in serotonin release, whereas others induce responses to persistent release. Whole-brain calcium imaging reveals widespread serotonin-associated brain dynamics, impacting different behavioral networks in different ways. We map out all sites of serotonin receptor expression in the connectome, which, together with synaptic connectivity, helps predict serotonin-associated brain-wide activity changes. These results provide a global view of how serotonin acts at defined sites across a connectome to modulate brain-wide activity and behavior.
A scoping review of trauma informed approaches in acute, crisis, emergency and residential mental health care
Authors: Saunders Katherine R. K. , McGuinness Elizabeth , Barnett Phoebe , Foye Una , Sears Jessica , Carlisle Sophie , Allman Felicity , Tzouvara Vasiliki , Schlief Merle , San Juan Norha Vera , Stuart Ruth , Griffiths Jessica , Appleton Rebecca , McCrone Paul , Olive Rachel Rowan , Nyikavaranda Patrick , Jeynes Tamar , Mitchell Lizzie , Simpson Alan , Johnson Sonia , Trevillion Kylee
Published: January 2023

Experiences of trauma in childhood and adulthood are highly prevalent among service users accessing acute, crisis, emergency, and residential mental health services. These settings, and restraint and seclusion practices used, can be extremely traumatic, leading to a growing awareness for the need for trauma informed care (TIC). The aim of TIC is to acknowledge the prevalence and impact of trauma and create a safe environment to prevent re-traumatisation. This scoping review maps the TIC approaches delivered in these settings and reports related service user and staff experiences and attitudes, staff wellbeing, and service use outcomes.

We searched seven databases (EMBASE; PsycINFO; MEDLINE; Web of Science; Social Policy and Practice; Maternity and Infant Care Database; Cochrane Library Trials Register) between 24/02/2022-10/03/2022, used backwards and forwards citation tracking, and consulted academic and lived experience experts, identifying 4244 potentially relevant studies. Thirty-one studies were included.

Most studies (n=23) were conducted in the USA and were based in acute mental health services (n=16). We identified few trials, limiting inferences that can be drawn from the findings. The Six Core Strategies (n=7) and the Sanctuary Model (n=6) were the most commonly reported approaches. Rates of restraint and seclusion reportedly decreased. Some service users reported feeling trusted and cared for, while staff reported feeling empathy for service users and having a greater understanding of trauma. Staff reported needing training to deliver TIC effectively.

TIC principles should be at the core of all mental health service delivery. Implementing TIC approaches may integrate best practice into mental health care, although significant time and financial resources are required to implement organisational change at scale. Most evidence is preliminary in nature, and confined to acute and residential services, with little evidence on community crisis or emergency services. Clinical and research developments should prioritise lived experience expertise in addressing these gaps.
The central nervous system’s proteogenomic and spatial imprint upon systemic viral infections with SARS-CoV-2
Authors: Radke Josefine , Meinhardt Jenny , Aschman Tom , Chua Robert Lorenz , Farztdinov Vadim , Lukkassen Sören , Ten Foo Wei , Friebel Ekaterina , Ishaque Naveed , Franz Jonas , Huhle Valerie Helena , Mothes Ronja , Peters Kristin , Thomas Carolina , Streit Simon , Manitius Regina von , Körtvélyessy Péter , Vielhaber Stefan , Reinhold Dirk , Hauser Anja , Osterloh Anja , Enghard Philipp , Ihlow Jana , Elezkurtaj Sefer , Horst David , Kurth Florian , Müller Marcel A. , Gassen Nils C. , Schneider Julia , Jechow Katharina , Timmermann Bernd , Fernandez-Zapata Camila , Böttcher Chotima , Stenzel Werner , Wyler Emanuel , Corman Victor , Stadelmann-Nessler Christine , Ralser Markus , Eils Roland , Heppner Frank L. , Mülleder Michael , Conrad Christian , Radbruch Helena
Published: January 2023

In COVID-19 neurological alterations are noticed during the systemic viral infection. Various pathophysiological mechanisms on the central nervous system (CNS) have been suggested in the past two years, including the viral neurotropism hypothesis. Nevertheless, neurological complications can also occur independent of neurotropism and at different stages of the disease and may be persistent.

Previous autopsy studies of the CNS from patients with severe COVID-19 show infiltration of macrophages and T lymphocytes, especially in the perivascular regions as well as pronounced microglial activation, but without signs of viral encephalitis.

However, there is an ongoing debate about long-term changes and cytotoxic effects in the CNS due to the systemic inflammation.

Here, we show the brain-specific host response during and after COVID-19. We profile single-nucleus transcriptomes and proteomes of brainstem tissue from deceased COVID-19 patients who underwent rapid autopsy. We detect a disease phase-dependent inflammatory type-I interferon response in acute COVID-19 cases. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation. One neuronal with direct focus on cranial nerve nuclei and one diffusely affecting the whole brainstem, the latter reflecting a bystander effect that spreads throughout the vascular unit and alters the transcriptional state of oligodendrocytes, microglia and astrocytes.

Our results indicate that even without persistence of SARS-CoV-2 in the CNS, the tissue activates highly protective mechanisms, which also cause functional disturbances that may explain the neurological symptoms of COVID-19, triggered by strong systemic type-I IFN signatures in the periphery.
Patterns of infection among travellers to Singapore arriving from mainland China
Authors: Pung Rachael , Kucharski Adam J. , Marc Ho Zheng Jie , Lee Vernon J.
Published: January 2023

In light of the rapid growth of COVID-19 in mainland China, countries and regions outside of China have implemented travel restrictions of varying intensity. Using surveillance data of symptomatic travellers arriving from mainland China and detected in Singapore, this provides a proxy on the COVID-19 in mainland China. Furthermore, this allows us to ensure that travel-related restrictions commensurate with the current epidemiological situation and risk.
High risk injection drug use and uptake of HIV prevention and treatment services among people who inject drugs in KwaZulu-Natal, South Africa
Authors: Zanoni Brian C. , Milford Cecilia , Sithole Kedibone , Mosery Nzwakie , Wilson Michael , Bosman Shannon , Smit Jennifer
Published: January 2023

The use of injection drugs in South Africa is increasing. HIV prevention, treatment and addiction services for people who inject drugs (PWID) in South Africa are not well documented. We conducted a mixed-methods study to understand current drug use practices and access to HIV prevention and treatment services for PWID in KwaZulu-Natal, South Africa. We used respondent-driven sampling to recruit 45 people who reported injecting opiates within the past 6 months from Durban, KwaZulu-Natal, South Africa. We found high rates of practices that increase HIV/viral hepatitis risk including the use of shared needles (43%) and direct blood injections (bluetoothing) (18%). Despite 35% of participants living with HIV, only 40% accessed antiretroviral therapy within the past year, and one accessed PrEP. None of the participants reported ever testing for Hepatitis C.
Listract test: a Standardized Assessment Method for Isolated Lisfranc Instability in Cadaver Models
Authors: Mathur Vasundhara , Osei-Hwedieh David O. , Sayyed Hosseinian Sayyed Hadi , Raduan Fernando , Kaiser Philip , Ghaednia Hamid , Waryasz Gregory , Bejarano-Pineda Lorena , Kwon John , Ashkani-Esfahani Soheil
Published: January 2023

Background

The gold standard for diagnosis of Lisfranc instability is direct visualization in the operation room while the examination techniques is still unstandardized and non-reproducible. We aimed to introduce a novel reproducible intraoperative mechanical testing method (Listract test) for intraoperative isolated Lisfranc instability assessment.

Methods

The Lisfranc ligament between the first cuneiform (C1) and second metatarsus (M2) in eight lower leg cadaveric specimens were dissected to replicate C1-M2 Lisfranc instability by eight foot and ankle surgeons. The 50N distraction force was applied in the direction of the C1-M2 ligament. Three methods of fixation - flexible fixation, metal screw, and bio-absorbable screw- were used to fix the injury, and Listract test was applied again after fixation. Besides intraoperative assessment, C1-M2 diastasis and area were measured using radiographs for assessment of Lisfranc instability.

Results

The sensitivity and specificity of the Listract test for detection of C1-M2 instability were 100% and 100% intraoperatively, 33.3% and 95.2% using radiographic diastasis measurement, and 63.2% and 38% using area measurement, respectively. The Listract test had a specificity and sensitivity of 100% and 96% for intraoperative assessment, 87.5% and 64.3 for radiographic C1-M2 diastasis, and 48% and 50% for radiographic area.

Conclusion

The Listract test is a simple, reproducible, and replicable intraoperative method for evaluating the Lisfranc joint for instability. Developing a device with this mechanism can help clinicians confirm the diagnosis and provide appropriate treatment particularly for equivocal diagnoses.

Authors’ contributions

SAE: Conception, design, and conducting the study, analysis, drafting the manuscript; VM, DOH, SHSH, FR: conducting the study, analysis, preparing the manuscript draft; LBP, JK, GW, PK: conducting the study, manuscript preparation, discussion.
Nr5a2is essential for morula development
Authors: Festuccia Nicola , Vandormael-Pournin Sandrine , Chervova Almira , Geiselman Anna , Langa-Vives Francina , Coux Rémi-Xavier , Gonzalez Inma , Cohen-Tannoudji Michel , Navarro Pablo
Published: January 2023

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. While the TFs specifying the blastocyst’s lineages are well characterised, those playing earlier roles are ill-defined. Using mouse models of the TFNr5a2, we show thatNr5a2-/-embryos arrest at the early morula stage and exhibit overt phenotypical problems such as altered lineage specification, frequent mitotic failure and substantial chromosome segregation defects. Transcriptomic profiling shows that NR5A2 is a master regulator required for appropriate expression of thousands of genes at the 8-cells stage, including lineage-specifying TFs and genes involved in mitosis, telomere maintenance and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability and lineage specification to ensure proper morula development.
Ontogenesis of the molecular response to sleep loss
Authors: Muheim Christine M. , Ford Kaitlyn , Medina Elizabeth , Singletary Kristan , Peixoto Lucia , Frank Marcos G.
Published: January 2023

Sleep deprivation (SD) results in profound cellular and molecular changes in the adult mammalian brain. Some of these changes may result in, or aggravate, brain disease. However, little is known about how SD impacts gene expression in developing animals. We examined the transcriptional response in the prefrontal cortex (PFC) to SD across postnatal development in male mice. We used RNA sequencing to identify functional gene categories that were specifically impacted by SD. We find that SD has dramatically different effects on PFC genes depending on developmental age. Gene expression differences after SD fall into 3 categories: present at all ages (conserved), present when mature sleep homeostasis is first emerging, and those unique to certain ages in adults. Developmentally conserved gene expression was limited to a few functional categories, including Wnt-signaling which suggests that this pathway is a core mechanism regulated by sleep. In younger ages, genes primarily related to growth and development are affected while changes in genes related to metabolism are specific to the effect of SD in adults.
Experimental Tests of the Virtual Circular Genome Model for Non-enzymatic RNA Replication
Authors: Ding Dian , Zhou Lijun , Mittal Shriyaa , Szostak Jack W.
Published: January 2023

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extension on a subset of kinetically trapped partially base-paired configurations, followed by shuffling of these configurations to enable continued oligonucleotide elongation. Here we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of length 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides, and investigated factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system, since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is pre-activated. We discuss the necessity of ongoingin-situactivation chemistry for continuous and accurate VCG replication.
Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes in acute myeloid leukemia
Authors: Chun Changju , Byun Ja Min , Cha Minkwon , Lee Hongwon , Choi Byungsan , Kim Hyunwoo , Hong Saem , Lee Yunseo , Park Hayoung , Koh Youngil , Yoon Tae-Young
Published: January 2023

BH3 mimetics are protein-protein interaction (PPI) inhibitors that saturate anti-apoptotic proteins in the BCL2 family to induce apoptosis in cancer cells, a prominent example of protein complex-targeting therapies. Despite the remarkable success of the BH3 mimetic ABT-199 in treating hematological malignancies, only a fraction of patients respond to ABT-199 and eventually develop resistance, necessitating the predictive biomarkers for both initial responses and resistance development. We here used the single-molecule pull-down and co-immunoprecipitation platform to quantify more than 20 different types of PPI complexes using ∼1.2×106cells in total, revealing the rewired status of the BCL2 family PPI network. By comparing the obtained multi-dimensional data with BH3 mimetic efficacies determinedex vivo, we constructed an analysis model for ABT-199 efficacy that designates the BCL2-BAX and BCLxL-BAK complexes as the primary mediators of drug effectiveness and resistance. We then applied this model to assist in therapeutic decision-making for acute myeloid leukemia patients in a prospective manner. Our work demonstrates a capability for the extensive characterization of PPI complexes in clinical specimens, potentially opening a new avenue of precision medicine for protein complex-targeting therapies.
Mental health services implementation in Colombia – a systematic review
Authors: Alarcón Garavito Germán Andrés , Burgess Rochelle , Dedios Sanguinetti María Cecilia , Peters Laura E.R. , San Juan Norha Vera
Published: January 2023

Colombia’s mental health services have a complex history shaped by 60 years of armed conflict, a predominantly clinical approach to mental health, and social factors such as inequities and stigma. The 1990 Caracas declaration proposed a shift towards decentralised community mental health services and interventions based on the recovery approach and emphasis on social determinants of mental health in the Americas. Colombia has adopted these approaches in its legal and practical framework in recent years, but implementation has been uneven. This systematic review aims to contribute to mental health services understanding in Colombia by reviewing published studies about mental health services and interventions.

A search was conducted to explore peer-reviewed studies on Colombian mental health services across five databases (Medline, PubMed, Scopus, Scielo and BVS [Biblioteca Virtual de Salud in Spanish]) on papers published in the last ten years and without language restrictions. The Consolidated Framework for Implementation Research (CFIR) was used to structure the analysis and identify barriers and facilitators during the implementation of mental health services. We adapted the CFIR to attend to gender, race and age informed by the Socio-Political Economy of Global Mental Health framework, given the importance of these factors to the Colombian health landscape.

1 530 records were identified, and 12 articles met all inclusion criteria and were included in the analysis. 8 papers described substance use disorders services, 11 involved multidisciplinary healthcare professionals, and 7 were implemented at a local scale. The primary barriers to implementation were the lack of coordination, high workloads, and low funding. Facilitators included the use of protocols, and the involvement of communities, stakeholders, users, and external champions.

Findings suggest the continued importance of community and recovery approaches and efforts to improve coordination between multi-sector actors involved in the mental health spaces (e.g., public, and private organisations, users and their families).
“Climate change and health?”: Knowledge and perceptions among key stakeholders in Puducherry, India
Authors: Shrikhande Shreya S , Merten Sonja , Cambaco Olga , Lee Tristan , Lakshmanasamy Ravivarman , Röösli Martin , Dalvie Mohammad Aqiel , Utzinger Jürg , Cissé Guéladio
Published: January 2023

Introduction

Climate change has far-reaching impacts on human health, which often differ by contextual vulnerabilities and region, with low-and middle income-countries being particularly vulnerable. Recognizing this, India has launched regional climate change and health action plans, the development, strengthening and implementation of which partially depends on how well stakeholders understand the topic. As little is known about this topic, our study aimed to better understand the knowledge and perceptions on the impacts of climate change on health amongst key stakeholders in Puducherry, India.

Methods

We conducted a qualitative study employing key informant interviews with medical doctors, researchers, environmentalists and governmental officials working on the climate change agenda from Puducherry. A total of 16 interviews were conducted. Half of the participants were practicing medical doctors while the other half consisted of medical researchers and government officials. The findings were analysed using the framework method, which allowed for data driven thematic analysis.

Results

We elucidated that despite elaborating the direct and indirect impacts of climate change on health, there is insufficient education and knowledge about the topic among participants. Knowledge of the public health burden and vulnerabilities influenced the perceived health risks from climate change, with some level of scepticism on the impacts on non-communicable diseases, such as cardiovascular diseases. There was also a felt need for multi-level awareness and intervention programs targeting all societal levels along with recommendations for educational changes and adaptation measures in the region.

Conclusion

This study identified a gap in the knowledge and understanding of health impacts of climate change among key stakeholders in Puducherry. It also identified region specific vulnerabilities that can be taken into consideration for strengthening the region’s climate change and health adaptation policy. In light of limited research on this topic, especially in a developing settings, our study lends to an improved understanding of how key stakeholders perceive the impacts of climate change on health.
DksA, ppGpp and RegAB regulate nitrate respiration inParacoccus denitrificans
Authors: Ray Ashvini , Spiro Stephen
Published: January 2023

The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases ofParacoccus denitrificansare responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here we show that deletion of eitherdksA1(encoding one of twodksAhomologs in theP. denitrificansgenome) orrelA/spoT(encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase innappromoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation thatnappromoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. ThedksA1andrelA/spoTgenes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the seconddksAhomolog (dksA2) andrelA/spoThave roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation.

IMPORTANCE

Paracoccus denitrificansis a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.
Knocking out alpha-synuclein in melanoma cells downregulates L1CAM and decreases motility
Authors: Gajendran Nithya , Rajasekaran Santhanasabapathy , Witt Stephan N.
Published: January 2023

The Parkinson’s disease (PD) associated protein, alpha-synuclein (α-syn/SNCA), is highly expressed in aggressive melanomas, which raises the possibility that α-syn has a pro-survival function in melanoma. Herein, we asked whether α-syn modulates the expression of the pro-oncogenic adhesion molecules L1CAM and N-cadherin. We used two human melanoma cell lines (SK-MEL-28, SK-MEL-29),SNCA-knockout (KO) clones, and two human SH-SY5Y neuroblastoma cell lines. In the melanoma lines, loss of α-syn expression resulted in significant decreases in the expression of L1CAM and N-cadherin and concomitant significant decreases in motility. On average, there was a 75% reduction in motility in the fourSNCA-KOs tested compared to control cells. Strikingly, comparing neuroblastoma SH-SY5Y cells that have no detectable α-syn to SH-SY5Y cells that stably express α-syn (SH/+αS), we found that expressing α-syn increased L1CAM and single-cell motility by 54% and 597%, respectively. The reduction in L1CAM level inSNCA-KO clones was not due to a transcriptional effect, rather we found that L1CAM is more efficiently degraded in the lysosome inSNCA-KO clones than in control cells. We propose that α-syn is pro-survival to melanoma (and possibly neuroblastoma) because it promotes the intracellular trafficking of L1CAM.
RepairSwitch: simultaneous functional assessment of homologous recombination vs end joining DNA repair pathways in living cells
Authors: Steinberg Ruchama C. , Liu Jianyong , Vaghasia Ajay M. , Giovinazzo Hugh , Pham Minh-Tam , Tselenchuk Dimitri , Chikarmane Roshan , Haffner Michael C. , Nelson William G. , Yegnasubramanian Srinivasan
Published: January 2023

DNA repair pathways are frequently defective in human cancers. DNA double strand breaks (DSBs) are most often repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ). Alterations in repair pathways can indicate sensitivity to therapeutic agents such as PARP inhibitors, cisplatin, and immunotherapy. Thus, functional assays to measure rates of HR and NHEJ are of significant interest. Several methods have been developed to measure rates of HR or NHEJ; however, there is a need for functional cell-based assays that can measure rates by both major DNA DSB pathways simultaneously. Here, we describe the RepairSwitch assay, a flow cytometry assay to assess rates of HR and NHEJ mediated repair of Cas9 programmed DSB simultaneously using a novel fluorescence switching reporter system. The assay exhibits low background signal and is capable of detecting rare repair events in the 1 in 10,000 range. We demonstrate the utility of RepairSwitch by measuring the potency of inhibitors of ATM (KU-60019, KU-55933), DNA-PK (NU7441), and PARP (Olaparib) on modulating DSB repair rates in HEK293FT cells. The selective ATM inhibitor KU-60019 inhibited HR rates with IC50 of 915 nM. Interestingly, KU-60019 exposure led to a dose responsive increase in rates of NHEJ. In contrast, the less selective ATM inhibitor KU-55933, which also has activity on DNA-PK, showed inhibition of both HR and NHEJ. The selective DNA-PK inhibitor NU7441 inhibited NHEJ efficiency with an IC50 of 299 nM, and showed a dose responsive increase in HR. The PARP inhibitor Olaparib showed lower potency in modulating HR and NHEJ. We next used the RepairSwitch assay to assess how pharmacological and genetic inhibition of DNA methyltransferases (DNMT) impacted rates of HR and NHEJ. The DNMT inhibitor decitabine reduced HR, but increased rates of NHEJ, both in a dose responsive manner, in both HEK293FT and HCT116 cells (IC50 for HR of 187 nM and 1.4 uM respectively). Knockout of DNMT1 and DNMT3B increased NHEJ, while knockout of DNMT3B, but not DNMT1, reduced HR. These results illustrate the utility of RepairSwitch as a functional assay for measuring changes in rates of DSB repair induced by pharmacological or genetic perturbation. Furthermore, the findings illustrate the potential for one DNA repair mechanism to compensate in part for loss of another. Finally, we showed that inhibition of DNMT can lead to reduction of HR and increase in NHEJ, providing some additional insight into recently observed synergy of DNMT inhibitors with PARP inhibitors for cancer treatment.
Genomic Landscape of Patients with GermlineRUNX1Variants and Familial Platelet Disorder with Myeloid Malignancy
Authors: Yu Kai , Deuitch Natalie , Merguerian Matthew , Cunningham Lea , Davis Joie , Bresciani Erica , Diemer Jamie , Andrews Elizabeth , Young Alice , Donovan Frank , Sood Raman , Craft Kathleen , Chong Shawn , Chandrasekharappa Settara , Mullikin Jim , Liu Paul P.
Published: January 2023

GermlineRUNX1mutations lead to familial platelet disorder with associated myeloid malignancies (FPDMM), which is characterized by thrombocytopenia and a life-long risk (35-45%) of hematological malignancies. We recently launched a longitudinal natural history study for patients with FPDMM at the NIH Clinical Center. Among 29 families with research genomic data, 28 different germlineRUNX1variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 24 of 54 (44.4%) non-malignant patients, somatic mutations were detected in at least one of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes.BCORwas the most frequently mutated gene (in 9 patients), and multipleBCORmutations were identified in 4 patients. Mutations in 7 other CHIP or AML driver genes (DNMT3A, TET2, NRAS, SETBP1, SF3B1, KMT2C, andLRP1B) were also found in more than one non-malignant patient. Moreover, three unrelated patients (one with myeloid malignancy) carried somatic mutations inNFE2, which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in elderly patients. In summary, there are diverse types of germlineRUNX1mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring dynamic changes of somatic mutations prospectively will benefit patients’ clinical management and reveal mechanisms for progression to myeloid malignancies.

Key Points

Comprehensive genomic profile of patients with FPDMM with germlineRUNX1mutations.

Rising clonal hematopoiesis related secondary mutations that may lead to myeloid malignancies.
Adaptive optical two-photon fluorescence microscopy probes cellular organization of ocular lenses in vivo
Authors: Paidi Santosh Kumar , Zhang Qinrong , Yang Yuhan , Xia Chun-Hong , Ji Na , Gong Xiaohua
Published: January 2023

The mammalian ocular lens is an avascular multicellular organ that grows continuously throughout life. Traditionally, its cellular organization is investigated using dissected lenses, which eliminates in vivo environmental and structural support. Here, we demonstrated that two-photon fluorescence microscopy (2PFM) can visualize lens cells in vivo. To maintain subcellular resolution at depth, we employed adaptive optics (AO) to correct aberrations due to ocular and lens tissues, which led to substantial signal and resolution improvements. Imaging lens cells up to 980 μm deep, we observed novel cellular organizations including suture-associated voids, enlarged vacuoles, and large cavities, contrary to the conventional view of a highly ordered organization. We tracked these features longitudinally over weeks and observed the incorporation of new cells during growth. Taken together, non-invasive longitudinal in vivo imaging of lens morphology using AO 2PFM will allow us to directly observe the development or alterations of lens cellular organization in living animals.
Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology
Authors: Aguado Julio , Amarilla Alberto A. , Taherian Fard Atefeh , Albornoz Eduardo A. , Tyshkovskiy Alexander , Schwabenland Marius , Chaggar Harman K. , Modhiran Naphak , Gómez-Inclán Cecilia , Javed Ibrahim , Baradar Alireza A. , Liang Benjamin , Dharmaratne Malindrie , Pietrogrande Giovanni , Padmanabhan Pranesh , Freney Morgan E. , Parry Rhys , Sng Julian D.J. , Isaacs Ariel , Khromykh Alexander A. , Rojas-Fernandez Alejandro , Davis Thomas P. , Prinz Marco , Bengsch Bertram , Gladyshev Vadim N. , Woodruff Trent M. , Mar Jessica C. , Watterson Daniel , Wolvetang Ernst J.
Published: January 2023

Aging is the primary risk factor for most neurodegenerative diseases, and recently coronavirus disease 2019 (COVID-19) has been associated with severe neurological manifestations that can eventually impact neurodegenerative conditions in the long-term. The progressive accumulation of senescent cellsin vivostrongly contributes to brain aging and neurodegenerative co-morbidities but the impact of virus-induced senescence in the aetiology of neuropathologies is unknown. Here, we show that senescent cells accumulate in physiologically aged brain organoids of human origin and that senolytic treatment reduces inflammation and cellular senescence; for which we found that combined treatment with the senolytic drugs dasatinib and quercetin rejuvenates transcriptomic human brain aging clocks. We further interrogated brain frontal cortex regions in postmortem patients who succumbed to severe COVID-19 and observed increased accumulation of senescent cells as compared to age-matched control brains from non-COVID-affected individuals. Moreover, we show that exposure of human brain organoids to SARS-CoV-2 evoked cellular senescence, and that spatial transcriptomic sequencing of virus-induced senescent cells identified a unique SARS-CoV-2 variant-specific inflammatory signature that is different from endogenous naturally-emerging senescent cells. Importantly, following SARS-CoV-2 infection of human brain organoids, treatment with senolytics blocked viral retention and prevented the emergence of senescent corticothalamic and GABAergic neurons. Furthermore, we demonstrate in human ACE2 overexpressing mice that senolytic treatment ameliorates COVID-19 brain pathology following infection with SARS-CoV-2.In vivotreatment with senolytics improved SARS-CoV-2 clinical phenotype and survival, alleviated brain senescence and reactive astrogliosis, promoted survival of dopaminergic neurons, and reduced viral and senescence-associated secretory phenotype gene expression in the brain. Collectively, our findings demonstrate SARS-CoV-2 can trigger cellular senescence in the brain, and that senolytic therapy mitigates senescence-driven brain aging and multiple neuropathological sequelae caused by neurotropic viruses, including SARS-CoV-2.
Establishment of Wnt ligand-receptor organization and cell polarity in theC. elegansembryo
Authors: Recouvreux Pierre , Pai Pritha , Torro Rémy , Ludányi Mónika , Mélénec Pauline , Boughzala Mariem , Bertrand Vincent , Lenne Pierre-François
Published: January 2023

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported and detected. The spatiotemporal dynamics of signaling molecules is largely unknown, yet it determines signal activity’s range and time frame. Here, we use theCaenorhabditis elegansembryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how locally produced Wnt ligands spread to transmit information to distant target cells. With quantitative live imaging, we show that the Wnt ligands diffuse extracellularly through the embryo over a timescale shorter than the cell cycle. We extract diffusion coefficients of Wnt ligands and their receptor Frizzled (Fz) and characterize their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize target cells. Our results support diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.
GraphGPSM: a global scoring model for protein structure using graph neural networks
Authors: He Guangxing , Liu Jun , Liu Dong , Guijun Zhang
Published: January 2023

The scoring models used for protein structure modeling and ranking are mainly divided into unified field and protein-specific scoring functions. Although protein structure prediction has made tremendous progress since CASP14, the modeling accuracy still cannot meet the requirements to a certain extent. Especially, accurate modeling of multi-domain and orphan proteins remains a challenge. Therefore, an accurate and efficient protein scoring model should be developed urgently to guide the protein structure folding or ranking through deep learning. In this work, we propose a protein structure global scoring model based on equivariant graph neural network (EGNN), named GraphGPSM, to guide protein structure modeling and ranking. We construct an EGNN architecture, and a message passing mechanism is designed to update and transmit information between nodes and edges of the graph. Finally, the global score of the protein model is output through a multilayer perceptron. Residue-level ultrafast shape recognition is used to describe the relationship between residues and the overall structure topology, and distance and direction encoded by Gaussian radial basis functions are designed to represent the overall topology of the protein backbone. These two features are combined with Rosetta energy terms, backbone dihedral angles, and inter-residue distance and orientations to represent the protein model and embedded into the nodes and edges of the graph neural network. The experimental results on the CASP13, CASP14, and CAMEO test sets show that the scores of our developed GraphGPSM have a strong correlation with the TM-score of the models, which are significantly better than those of the unified field score function REF2015 and the state-of-the-art local lDDT-based scoring models ModFOLD8, ProQ3D, and DeepAccNet etc. The modeling experimental results on 484 test proteins demonstrate that GraphGPSM can greatly improve the modeling accuracy. GraphGPSM is further used to model 35 orphan proteins and 57 multi-domain proteins. The results show that the average TM-score of the models predicted by GraphGPSM is 13.2% and 7.1% higher than that of the models predicted by AlphaFold2. GraphGPSM also participates in CASP15 and achieves competitive performance in global accuracy estimation.
Bayesian Differential Analysis of Cell Type Proportions
Authors: Karagiannis Tanya T. , Monti Stefano , Sebastiani Paola
Published: January 2023

The analysis of cell type proportions in a biological sample should account for the compositional nature of the data but most analyses ignore this characteristic with the risk of producing misleading conclusions. The recent method scCODA appropriately incorporates these constraints by using a Bayesian Multinomial-Dirichlet model that requires a reference cell type to normalize the distribution of all cell types. However, a reference cell type that is stable across biological conditions may not always be available. Here, we present an approach that uses a Bayesian multinomial regression for the analysis of single cell distribution data without the need for a reference cell type. We show an implementation example using the rjags package within the R software.
Molecular Imaging with Fibroblast Activation Protein Tracers depicts Inflammatory Joint Damage and its Transition to Resolution of Inflammation
Authors: Rauber Simon , Mohammadian Hashem , Schmidkonz Christian , Atzinger Armin , Soare Alina , Maschauer Simone , Treutlein Christoph , Angeli Mario , Raimondo Maria Gabriella , Xu Cong , Yang Kai-Ting , Lu Le , Labinsky Hannah , Kampylafka Eleni , Knitza Johannes , Maric Hans , Distler Jörg H.W. , Bäuerle Tobias , Kuwert Torsten , Prante Olaf , Cañete Juan , Schett Georg , Ramming Andreas
Published: January 2023

Joint fibroblasts play an important role in the transition from joint inflammation to irreversible joint damage. There is no established clinical method to measure fibroblast activation during inflammation and their phenotypic dynamics upon therapy to date. Here we show that upon treatment with IL-17A/TNF-blocking antibodies fibroblasts change their phenotype from a destructive IL-6+/MMP3+THY1+to a CD200+DKK3+subtype, actively inducing resolution of inflammation. This phenotypic switch can be visualized due to so far unexplored different capacities of fibroblast subtypes with regard to receptor internalization of small molecular tracers with high affinity to FAP. Although FAP expression levels are comparable between fibroblast subtypes in the joint, FAP internalisation rate correlates with the destructive potential of fibroblasts and resolving fibroblasts have a lower FAP internalisation rate, providing a valuable imaging tool to visualize the transition from joint damage to resolution of inflammation.
GEARBOCS: An Adeno Associated Virus Tool forIn VivoGene Editing in Astrocytes
Authors: Sivadasan Bindu Dhanesh , Savage Justin T. , Brose Nicholas , Bradley Luke , Dimond Kylie , Tan Christabel Xin , Eroglu Cagla
Published: January 2023

CRISPR/Cas9-based genome engineering enables rapid and precise gene manipulations in the CNS. Here, we developed a non-invasive astrocyte-specific method utilizing a single AAV vector, which we named GEARBOCS (Gene Editing in AstRocytes Based On CRISPR/Cas9 System). We verified GEARBOCS’ specificity to mouse cortical astrocytes and demonstrated its utility for three types of gene manipulations: knockout (KO); tagging (TagIn); and reporter knock-in (GeneTrap) strategies. Next, we deployed GEARBOCS in two test cases. First, we determined that astrocytes are a necessary source of the synaptogenic factor Sparcl1 for thalamocortical synapse maintenance in the mouse primary visual cortex. Second, we determined that cortical astrocytes express the synaptic vesicle associated Vamp2 protein and found that it is required for maintaining excitatory and inhibitory synapse numbers in the visual cortex. These results show that the GEARBOCS strategy provides a fast and efficient means to study astrocyte biologyin vivo.

Motivation

Astrocytes are indispensable for brain development, function, and health. However, molecular tools to study astrocyte biology and functionin vivohave been largely limited to genetically modified mice. Here, we developed a CRISPR/Cas9-based gene editing strategy within a single AAV vector that enables efficient genome manipulations in astrocytes. We designed and optimized this easy-to-use viral tool to understand gene expression, protein localization and function in astrocytesin vivo.
Metacognitive Judgments during Visuomotor Learning Reflect the Integration of Error History
Authors: Hewitson Christopher L. , Al-Fawakhiri Naser , Forrence Alexander D. , McDougle Samuel D.
Published: January 2023

People form metacognitive representations of their own abilities across a range of tasks. How these representations are influenced by errors during learning is poorly understood. Here we ask how metacognitive confidence judgments of performance during motor learning are shaped by the learner’s recent history of errors. Across four motor learning experiments, our computational modeling approach demonstrated that people’s confidence judgments are best explained by a recency-weighted averaging of visually observed errors. Moreover, in the formation of these confidence estimates, people appear to re-weight observed motor errors according to a subjective cost function. Confidence judgments were adaptive, incorporating recent motor errors in a manner that was sensitive to the volatility of the learning environment, integrating a shallower history when the environment was more volatile. Finally, confidence tracked motor errors in the context of both implicit and explicit motor learning, but only showed evidence of influencing behavior in the latter. Our study thus provides a novel descriptive model that successfully approximates the dynamics of metacognitive judgments during motor learning.

NEW & NOTEWORTHY

This study examined how, during visuomotor-learning, people’s confidence in their performance is shaped by their recent history of errors. Using computational modeling, we found that confidence incorporated recent error-history, tracked subjective error-costs, was sensitive to environmental volatility, and in some contexts may influence learning. Together, these results provide a novel model of metacognitive judgments during motor-learning that could be applied to future computational and neural studies at the interface of higher-order cognition and motor control.