bluuebunny/crossref_metadata_2025 huggingface dataset:
Defatting of Human Livers during Long-Term ex situ Normothermic Perfusion. Novel Strategy to Rescue Discarded Organs for Transplantation
Authors: Sousa Da Silva Richard X. , Borrego Lucia Bautista , Lenggenhager Daniela , Huwyler Florian , Binz Jonas , Mancina Leandro , Breuer Eva , Wernlé Kendra , Hefti Max , Müller Matteo , Cunningham Leslie , De Oliveira Michelle L. , Petrowsky Henrik , Weber Achim , Dutkowski Philipp , Hoffmann Waldemar , Gupta Anurag , Tibbitt Mark , Humar Bostjan , Clavien Pierre-Alain
Published: July 2023

Objective:

To develop a protocol for the defatting of steatotic liver grafts during long-term ex situ normothermic machine perfusion.

Background:

Despite the alarming increase in donor organ shortage, the highly prevalent fatty liver grafts are often discarded due to the risk of primary non-function. Effective strategies preventing such outcome are currently lacking. An exciting new avenue is the introduction of ex situ normothermic machine perfusion (NMP), enabling a liver to remain fully functional for up to two weeks and providing a unique window of opportunity for defatting prior to transplantation.

Methods:

Over a 5-year period, 23 discarded liver grafts and 28 partial livers from our resection program were tested during ex situ NMP. The steatosis degree was determined on serial biopsies by expert pathologists, and triglyceride contents measured simultaneously.

Results:

Of 51 liver grafts, 20 were steatotic, with up to 85% macrovesicular steatosis, and were perfused for up to 12 days. 10 livers displayed marked (5 of which almost complete) loss of fat, while the other 10 did not respond to long-term perfusion. Successful defatting was related to prolonged perfusion, automated glucose control, circadian nutrition, and L-carnitine/fenofibrate supplementation. Pseudopeliotic steatosis and the associated activation of Kupffer/stellate cells were unexpected processes that might contribute to defatting. Synthetic and metabolic function remained preserved for most grafts until perfusion end.

Conclusion:

Ex situ long-term perfusion effectively reduces steatosis while preserving organ viability and may in the future allow transplantation of primarily unusable high-risk grafts, significantly increasing the number of organs available for transplantation.
DBScope: a versatile computational toolbox for the visualization and analysis of sensing data from Deep Brain Stimulation
Authors: Oliveira Andreia M. , Carvalho Eduardo , Barros Beatriz , Soares Carolina , Ferreira-Pinto Manuel , Vaz Rui , Aguiar Paulo
Published: July 2023

Deep brain stimulation (DBS) is a therapy applied in numerous neurologic diseases, yielding major improvements in quality of life. Current implantable devices can record brain activity (in the form of local field potentials) at the site of stimulation, providing a window into the neuropathological phenomena and the potential to improve clinical care. Data-driven therapies often rely on tools to process, visualize, and analyze the data. However, existing tools in DBS are yet to fully exploit the devices’ sensing capabilities. We aimed to develop a user-friendly open-source toolbox for the visualization and analysis of sensing data from DBS. Special attention was given to enrich this toolbox with features which would foster its usefulness in both clinical and research environments. We developed a standalone MATLAB-based toolbox, called DBScope, capable of parsing the data generated by current sensing devices and producing rich visualizations with clinical and research relevance. The toolbox can be operated in two ways: through a user interface, bypassing programming experience requirements; and, programmatically, acting as a library of functions, which can be further adapted to user needs. We provide a detailed description of the toolbox features and exemplify its application in two case studies. DBScope is an open-source toolbox that provides visualization and analytical tools for clinical and research use, paving the way towards the improvement of data-driven DBS therapies. Additional functionalities are being considered for future updates.
Interdisciplinary Inquiry via PanelGPT: Application to Explore Chatbot Application in Sports Rehabilitation
Authors: McBee Joseph C. , Han Daniel Y. , Liu Li , Ma Leah , Adjeroh Donald A. , Xu Dong , Hu Gangqing
Published: July 2023

Background

ChatGPT showcases exceptional conversational capabilities and extensive cross-disciplinary knowledge. In addition, it possesses the ability to perform multiple roles within a single chat session. This unique multi-role-playing feature positions ChatGPT as a promising tool to explore interdisciplinary subjects.

Objective

The study intended to guide ChatGPT for interdisciplinary exploration through simulated panel discussions. As a proof-of-concept, we employed this method to evaluate the advantages and challenges of using chatbots in sports rehabilitation.

Methods

We proposed a model termed PanelGPT to explore ChatGPTs’ knowledge graph on interdisciplinary topics through simulated panel discussions. Applied to “chatbots in sports rehabilitation”, ChatGPT role-played both the moderator and panelists, which included a physiotherapist, psychologist, nutritionist, AI expert, and an athlete. We act as the audience posed questions to the panel, with ChatGPT acting as both the panelists for responses and the moderator for hosting the discussion. We performed the simulation using the ChatGPT-4 model and evaluated the responses with existing literature and human expertise.

Results

Each simulation mimicked a real-life panel discussion: The moderator introduced the panel and posed opening/closing questions, to which all panelists responded. The experts engaged with each other to address inquiries from the audience, primarily from their respective fields of expertise. By tackling questions related to education, physiotherapy, physiology, nutrition, and ethical consideration, the discussion highlighted benefits such as 24/7 support, personalized advice, automated tracking, and reminders. It also emphasized the importance of user education and identified challenges such as limited interaction modes, inaccuracies in emotion-related advice, assurance on data privacy and security, transparency in data handling, and fairness in model training. The panelists reached a consensus that chatbots are designed to assist, not replace, human healthcare professionals in the rehabilitation process.

Conclusions

Compared to a typical conversation with ChatGPT, the multi-perspective approach of PanelGPT facilitates a comprehensive understanding of an interdisciplinary topic by integrating insights from experts with complementary knowledge. Beyond addressing the exemplified topic of chatbots in sports rehabilitation, the model can be adapted to tackle a wide array of interdisciplinary topics within educational, research, and healthcare settings.
Prevalence and characteristics of genetic disease in adult kidney stone formers
Authors: Anderegg Manuel A. , Olinger Eric G. , Bargagli Matteo , Geraghty Rob , Pohlmeier Lea , Nater Alexander , Bruggmann Rémy , Sayer John A. , Vogt Bruno , Schaller André , Fuster Daniel G.
Published: July 2023

Background

Molecular mechanisms of kidney stone formation remain unknown in most patients. Previous studies showed high heritability of nephrolithiasis, but data on prevalence and characteristics of genetic disease in unselected adults with nephrolithiasis are lacking.

Methods

We performed whole exome sequencing in 787 participants of the Bern Kidney Stone Registry, an unselected cohort of adults with ≥ 1 past kidney stone episode (KSF), and 114 non- stone-forming individuals (NKSF). A total of 34 established nephrolithiasis genes were analyzed and variants assessed according to ACMG criteria. Pathogenic (P) or likely pathogenic (LP) variants were considered diagnostic.

Results

Mean age of KSF was 47±15 years, and 18 % were first time KSF. A Mendelian kidney stone disease was present in 2.9% (23 of 787) of KSF. The most common genetic diagnoses were cystinuria (SLC3A1,SLC7A9; n=13), Vitamin D-24 hydroxylase deficiency (CYP24A1; n=5) and primary hyperoxaluria (AGXT, GRHPR, HOGA1; n=3). 8.1% (64 of 787) of KSF were monoallelic for LP/P variants predisposing to nephrolithiasis, most frequently inSLC34A1/A3orSLC9A3R1(n=37),CLDN16(n=8) andCYP24A1(n=8). KSF with Mendelian disease had a lower age at the first stone event (30±14 years vs. 36±14 years, p=0.003), were more likely to have cystine stones (23.4 % vs. 1.4 %) and less likely to have calcium oxalate monohydrates stones (31.9 % vs. 52.5 %) compared to KSF without genetic diagnosis. The phenotype of KSF with variants predisposing to nephrolithiasis was subtle and showed significant overlap with KSF without diagnostic variants. In NKSF, no Mendelian disease was detected, and LP/P variants were significantly less prevalent compared to KSF (1.8 % vs. 8.1%).

Conclusion

Mendelian disease is uncommon in unselected adult KSF, yet variants predisposing to nephrolithiasis are significantly enriched in adult KSF.
Emergency Medical Systemic frustration of Aggression and Violence in Conflict Encounters: An Evolved Grounded Theory
Authors: Rees Nigel , Hawkes Claire , Williams Lauren , Williams Julia , O’Meara Peter
Published: July 2023

Background

Emergency Medical Services (EMS) staff worldwide have long been at risk of encountering violence and aggression (V&A) at work, including threats, verbal, physical, and sexual assault, and on rare occasions, fatalities occur. Exposure to V&A can result in stress, fear, and burnout. This is an international problem and EMS employers, trade unions and others are working towards tackling it. This paper reports the results of a qualitative study that aimed to explore protecting EMS Staff from aggression and violence in conflict encounters

Methods

This study took place in 2022 in one UK ambulance service covering a population of three million people. Individual, one-to-one semi-structured interviews were conducted with EMS staff via a Voice over Internet Protocol VoIP (VoIP). Data were analysed through Evolved Grounded theory methodology.

Results

Ten EMS staff were interviewed, and the following categories emerged:Rusted, busted and inevitability of Violence & Aggression in EMS Environment, Tolerable or intolerable Violence & aggression in EMS, Gendered violence & aggression and Genderization in EMS, modifiable factors and harm reduction of Violence & aggression in EMS, Professional, ethical & clinical judgments of Violence & aggression in EMS, and Socio-cultural and system frustrations of Violence & aggression in EMS.The Basic Socal Process (BSP) that emerged was one ofEmergency Medical Systemic frustration of Aggression and Violence in Conflict Encounters.

Conclusion

V&A directed toward EMS staff is complex and our participants revealed how it has long been accepted and may be influenced by systemic frustration in EMS. Staff reported frustrations due to tiredness and in patients from delays in EMS response. Female EMS staff experienced sexual V&A by co-workers and patients, and these voices reverberate with female EMS staff internationally. Our study revealed how EMS has not effectively tackled V&A and many sociocultural constructs accepted within EMS. We call for the voices of our participants to be amplified internationally and for purposeful efforts to continue to be made in tackling this issue.
Revealing a hidden conducting state by manipulating the intracellular domains in KV10.1 exposes the coupling between two gating mechanisms
Authors: Abdelaziz Reham , Tomczak Adam P. , Neef Andreas , Pardo Luis A.
Published: July 2023

TheKCNHfamily of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains ofKCNHchannels play a crucial role in gating that is still only partly understood. UsingKCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features ofKCNHchannels gating.
PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation
Authors: Du Changlin , Dong Jiahui , Xu Chuanting , Jia Pengcheng , Cai Na , Wang Qi , Zhang Zhenming , Jiang Lingfeng , Jiang Wei , Feng Rui , Li Jun , Huang Cheng , Ma Taotao
Published: July 2023

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by herbal medicines. Previously, we found that proline–serine–threonine phosphatase interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) play important roles in kidney injury and immune defense, respectively; however, the mechanism of AAN regulation by PSTPIP2 and NETs remains unclear. We found that renal tubular epithelial cell (RTEC) apoptosis, neutrophil infiltration, and inflammatory factor and NET production were increased in a mouse model of AAN, while PSTPIP2 expression was low. Conditional knock-in of PSTPIP2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. In contrast, restoring normal PSTPIP2 expression promoted kidney injury.In vivo,the use of Ly6G-neutralizing antibody to remove neutrophils and peptidyl arginine deiminase 4 (PAD4) inhibitors to prevent NET formation reduced apoptosis, thereby alleviating kidney injury.In vitro, damaged RTECs released interleukin-19 (IL-19) via the PSTPIP2/nuclear factor (NF)-κB pathway and induced NET formation via the IL-20Rβ receptor. Concurrently, NETs promoted the apoptosis of damaged RTECs. PSTPIP2 affected NET formation by regulating IL-19 expression via inhibition of NF-κB pathway activation in RTECs, inhibiting their apoptosis and reducing kidney damage.
Gestational physical exercise prevents early-life behavioral impairments and normalizes dopamine D2 receptor expression in the frontal cortex in the offspring of a rat model of attention deficit hyperactivity disorder
Authors: Tosta Andréa , Fonseca Ariene S. , Messender Débora , Siqueira Priscila , Ferreira Sérgio T. , Lourenco Mychael V. , Pandolfo Pablo
Published: July 2023

Attention deficit hyperactivity disorder (ADHD) is characterized by inattention, hyperactivity and impulsivity, and develops most frequently during childhood and adolescence. Spontaneously hypertensive rats (SHR) are the most used experimental model for the study of ADHD. SHR exhibit behavioral impairments that recapitulate phenotypes observed in individuals with ADHD. SHR further develop dopaminergic hypofunction in frontostriatal circuits and an imbalance in dopamine and norepinephrine systems. Maternal physical exercise (e.g., swimming) during pregnancy has been shown to promote angiogenesis, neurogenesis, learning, and memory in the offspring of control rats. We investigated the impact of gestational swimming on behavioral and dopaminergic parameters in childhood (1-2 weeks of age) and adolescent (4-5 weeks of age) SHR and Wistar Kyoto rats (WKY), used as a control. Maternal gestational swimming resulted in a reversal of neurodevelopmental impairments in behavior, assessed by the righting reflex and olfactory recognition tests, in the offspring. Furthermore, during adolescence, SHRs from exercised dams exhibited reduced novelty seeking, an important behavioral trait in this developmental period. Finally, SHRs exhibited increased expression of dopamine transporter (DAT) and D2 receptors (D2R) in the frontal cortex. D2R expression was normalized in the frontal cortex of adolescent SHRs whose mothers were exercised. Results suggest that physical exercise during pregnancy could be an effective preventative strategy against ADHD-associated behavioral and neurochemical phenotypes in the offspring.

Graphical Abstract
Sympathetic NPY controls glucose homeostasis, cold tolerance, and cardiovascular functions in mice
Authors: Kumari Raniki , Pascalau Raluca , Wang Hui , Bajpayi Sheetal , Yurgel Maria , Quansah Kwaku , Hattar Samer , Tampakakis Emmanouil , Kuruvilla Rejji
Published: July 2023

Neuropeptide Y (NPY) is best known for its effects in the brain as an orexigenic and anxiolytic agent and in reducing energy expenditure. NPY is also co-expressed with Norepinephrine (NE) in sympathetic neurons. Although NPY is generally considered to modulate noradrenergic responses, its specific roles in autonomic physiology remain under-appreciated. Here, we show that sympathetic-derived NPY is essential for metabolic and cardiovascular regulation in mice. NPY and NE are co-expressed in 90% of prevertebral sympathetic neurons and only 43% of paravertebral neurons. NPY-expressing neurons primarily innervate blood vessels in peripheral organs. Sympathetic-specific deletion of NPY elicits pronounced metabolic and cardiovascular defects in mice, including reductions in insulin secretion, glucose tolerance, cold tolerance, pupil size, and an elevation in heart rate, while notably, however, basal blood pressure was unchanged. These findings provide new knowledge about target tissue-specific functions of NPY derived from sympathetic neurons and imply its potential involvement in metabolic and cardiovascular diseases.
A Bayesian Regularized and Anotation-Informed Integrative Analysis of Cognition (BRAINIAC)
Authors: Zablocki Rong , Xu Bohan , Fan Chun-Chieh , Thompson Wesley K.
Published: July 2023

1

Here we present a development of the novel Bayesian Regularized and Anotation-Informed Integrative Analysis of Cognition (BRAINIAC) model. BRAINIAC allows for both estimation of total variance explained by all features for a given cognitive phenotype, as well as a principled assessment of the impact of annotations on relative enrichment of features compared to others in terms of variance explained, without relying on a potentially unrealistic assumption of sparsity of brain-cognition associations. We apply the BRAINIAC model to resting state fMRI data from the Adolescent Brain Cognitive Development Study.
Discontinuation of long-acting reversible contraceptive methods and associated factors among reproductive-age women in Shashemane town, Oromia, Ethiopia
Authors: Letose Fikru , Tusa Alemtsehay , Sahlu Degemu , Miherite Yohannis
Published: July 2023

Background

The early termination of long-acting reversible contraceptives raises issues for the healthcare system and has the potential to affect public health. Long-acting reversible contraception is now more widely available and used, although a sizable percentage of women still stop using it. Therefore, this study aims to assess factors associated with the discontinuation of the long-acting reversible contraceptive method among female users of health facilities in Shashemane Town.

Methods

A facility-based cross-sectional study was done in Shashemane town among 410 study participants. The study participants were selected by using a systematic random sampling method. Data were collected by using structured interviewer-administered questionnaires and entered into epi-data version 4.6.0.2 and exported to SPSS version 25 for analysis. Bivariate and multivariable logistic regressions were used to examine the association between independent variables and discontinuation of the long-acting reversible contraceptives. The results were presented using the Odds Ratio at 95% CI. P < 0.05 ware used to dictate statistical significance.

Result

The overall prevalence of women who removed the long-acting reversible contraceptive method before the due date was 57.2%. Factors such as having an occupation as a housewife, desire to become pregnant, unwarned side effects, effectiveness, and dissatisfaction with the service provided were positively associated with discontinuation of the contraception.

Conclusion and Recommendation

The prevalence of the discontinuation of reversible long-acting contraceptives was high. Pre-insertion effective counseling about the benefits, follow-up care and management for side effects, and client reassurance are recommended.
Degradation and release of dissolved environmental RNAs from zebrafish cells
Authors: Xu Zhongneng
Published: July 2023

Environmental RNAs in water are gradually being applied in aquatic ecological surveys, water pollution monitoring, etc., but the current methods to detect environmental RNAs in water can mainly measure the RNAs in the filters that are used for filtering water samples, neglecting dissolved environmental RNAs in water. The sources and degradation profiles of dissolved environmental RNAs in water remain unknown. The present study was conducted to measure the permeability of extracted RNAs from zebrafish cells through filters, the degradation of extracted RNAs from zebrafish cells in tubes, and the release rate and degradation of dissolved environmental RNAs from living zebrafish cells and dying zebrafish cells, aiming to provide dynamic information from dissolved environmental RNAs in water. The results showed that there were no significant differences between the levels of extracted RNAs from zebrafish cells before filtration with 0.45 µm filters and those in the filtrates. The extracted RNAs from zebrafish cells degraded in water in the tubes, and after 2 months, more than 15% of RNAs in the groups of RNAs in water were still detected. The half-life of all the RNAs in the tubes was approximately 20∼43 days. During the 6-day experiment of the release and degradation of dissolved RNAs from living cells, an average of 4.1×10-4∼ 1.7×10-3pg dissolved RNAs (7.6×105∼ 3.2×106RNA bases) were secreted per cell per day into the liquid environment. During the 6-day experiment of the release and degradation of dissolved RNAs from dying cells, approximately 4.2 pg of dissolved RNAs released by a dying zebrafish cell in water could be detected. The dissolved environmental RNAs in water from zebrafish cells degraded faster in the presence of zebrafish cells: under the conditions without zebrafish cells, the average survival rate of the dissolved environmental RNAs in water per day was 98.4%/day; under the conditions with living zebrafish cells, the average survival rate per day was 49.7%/day; and under the conditions with dying zebrafish cells, the average survival rate per day was 34.9%/day. The estimated levels of dissolved environmental RNAs in water in fish tanks were too low to be detected by the current techniques. Although the methods in the present study need to be improved, this study may provide information to develop new ways to measure the dynamics of dissolved environmental RNAs in water and quantitatively analyze RNAs released into liquid environments of living and dying cells.
γδ T cell-mediated cytotoxicity against patient-derived healthy and cancer cervical organoids
Authors: Dong Junxue , Holthaus David , Peters Christian , Koster Stefanie , Ehsani Marzieh , Quevedo-Olmos Alvaro , Berger Hilmar , Zarobkiewicz Michal , Mangler Mandy , Gurumurthy Rajendra Kumar , Hedemann Nina , Chumduri Cindrilla , Kabelitz Dieter , Meyer Thomas F.
Published: July 2023

Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies.

In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent a cell population with significant therapeutic potential against both hematologic and solid tumours.

To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function ofin vitroexpanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids.

To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair.

In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novelin vitromodel to optimize innovative patient-specific immunotherapies for cervical cancer.
PMS1as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington’s disease
Authors: McLean Zachariah L. , Gao Dadi , Correia Kevin , Roy Jennie C. L. , Shibata Shota , Farnum Iris N. , Valdepenas-Mellor Zoe , Rapuru Manasa , Morini Elisabetta , Ruliera Jayla , Gillis Tammy , Lucente Diane , Kleinstiver Benjamin P. , Lee Jong-Min , MacDonald Marcy E. , Wheeler Vanessa C. , Pinto Ricardo Mouro , Gusella James F.
Published: July 2023

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon ofHTTthat lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that targetHTTto reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both atHTTand other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate ofHTTCAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair genePMS1, a known modifier of HD age-at-onset. Genome editing at specificHTTandPMS1sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon inPMS1, making splice modulation ofPMS1a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.
Polyamine transport inhibition and cisplatin synergistically enhance tumor control through oxidative stress in murine head and neck cancer models
Authors: Yassin-Kassab Abdulkader , Wang Nathaniel , Foley Jackson , Stewart Tracy Murray , Burns Mark R. , Casero Robert A. , Harbison R. Alex , Duvvuri Umamaheswar
Published: July 2023

Background

Surgery and/or platinum-based chemoradiation remain standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While these therapies are effective in a subset of patients, a substantial proportion experience recurrence or treatment resistance. As cisplatin mediates cytotoxicity through oxidative stress while polyamines play a role in redox regulation, we posited that combining cisplatin with polyamine transport inhibitor, AMXT-1501, would increase oxidative stress and tumor cell death in HNSCC cells.

Methods

Cell proliferation was measured in syngeneic mouse HNSCC cell lines treated with cisplatin ± AMXT-1501. Synergy was determined by administering cisplatin and AMXT-1501 at a ratio of 1:10 to cancer cellsin vitro. Cancer cells were transferred onto mouse flanks to test the efficacy of treatmentsin vivo. Reactive oxygen species (ROS) were measured. Cellular apoptosis was measured with flow cytometry using Annexin V/PI staining. High-performance liquid chromatography (HPLC) was used to quantify polyamines in cell lines. Cell viability and ROS were measured in the presence of exogenous cationic amino acids.

Results

The combination of cisplatin and AMXT-1501 synergizein vitroon HNSCC cell lines.In vivocombination treatment resulted in tumor growth inhibition greater than either treatment individually. The combination treatment increased ROS production and induced apoptotic cell death. HPLC revealed the synergistic mechanism was independent of intracellular polyamine levels. Supplementation of cationic amino acids partially rescued cancer cell viability and reduced ROS.

Conclusion

AMXT-1501 enhances the cytotoxic effects of cisplatinin vitroandin vivoin aggressive HNSCC cell lines through a polyamine-independent mechanism.
Predicting Relative Populations of Protein Conformations without a Physics Engine Using AlphaFold2
Authors: Monteiro da Silva Gabriel , Cui Jennifer Y , Dalgarno David C , Lisi George P , Rubenstein Brenda M
Published: July 2023

This paper presents a novel approach for predicting the relative populations of protein conformations using AlphaFold 2, an AI-powered method that has revolutionized biology by enabling the accurate prediction of protein structures. While AlphaFold 2 has shown exceptional accuracy and speed, it is designed to predict proteins' single ground state conformations and is limited in its ability to predict fold switching and the effects of mutations on conformational landscapes. Here, we demonstrate how AlphaFold 2 can directly predict the relative populations of different conformations of proteins and even accurately predict changes in those populations induced by mutations by subsampling multiple sequence alignments. We tested our method against NMR experiments on two proteins with drastically different amounts of available sequence data, Abl1 kinase and the granulocyte-macrophage colony-stimulating factor, and predicted their relative state populations with accuracies in excess of 80%. Our method offers a fast and cost-effective way to predict protein conformations and their relative populations at even single point mutation resolution, making it a useful tool for pharmacology, analyzing NMR data, and studying the effects of evolution.
Integrative analysis of DNA replication origins and ORC/MCM binding sites in human cells reveals a lack of overlap
Authors: Tian Mengxue , Wang Zhenjia , Su Zhangli , Shibata Etsuko , Shibata Yoshiyuki , Dutta Anindya , Zang Chongzhi
Published: July 2023

Based on experimentally determined average inter-origin distances of ∼100 kb, DNA replication initiates from ∼50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the Origin Recognition Complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and 5 ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ∼7.5 million union origins identified by all datasets, only 0.27% were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques (20,250 shared origins), suggesting extensive variability in origin usage and identification. 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF binding sites, G-quadruplex sites and activating histone marks, these overlaps are comparable or less than that of known Transcription Start Sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ∼13,000 reproducible ORC binding sites in human cancer cells, and only 4.5% were within 1 kb of the ∼11,000 union MCM2-7 binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast,S. cerevisiae. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.
A signature of platelet reactivity in CBC scattergrams reveals genetic predictors of thrombotic disease risk
Authors: Verdier Hippolyte , Thomas Patrick , Batista Joana , Kempster Carly , McKinney Harriet , Gleadall Nicholas , Danesh John , Mumford Andrew , Heemskerk Johan , Ouwehand Willem H. , Downes Kate , Astle William J. , Turro Ernest
Published: July 2023

Genetic studies of platelet reactivity (PR) phenotypes may identify novel antiplatelet drug targets. However, discoveries have been limited by small sample sizes (n<5,000) due to the complexity of measuring PR. We trained a model to predict PR from complete blood count (CBC) scattergrams. A GWAS of this phenotype in 29,806 blood donors identified 21 distinct associations implicating 20 genes, of which six have been identified previously. The effect size estimates were significantly correlated with estimates from a study of flow-cytometry measured PR and a study of a phenotype ofin vitrothrombus formation. A genetic score of PR built from the 21 variants was associated with myocardial infarction and pulmonary embolism. Mendelian randomisation analyses showed PR to be causally associated with the risks of coronary artery disease, stroke and venous thromboembolism. Our approach provides a blueprint for employing phenotype imputation to study the determinants of hard-to-measure but biologically important haematological traits.

Key points

Platelet reactivity can be predicted from scattergrams generated by haematology analysers of a type in widespread clinical use.

Genetic analysis ofpredictedplatelet reactivity reveals associations with the risks of thrombotic diseases, including stroke.
Differential expansion and retention patterns of LRR-RLK genes across plant evolution
Authors: Kileeg Zachary , Haldar Aparna , Khan Hasna , Qamar Arooj , Mott G. Adam
Published: July 2023

To maximize overall fitness, plants must accurately respond to a host of growth, developmental, and environmental signals throughout their life. Many of these internal and external signals are perceived by the leucine-rich repeat receptor-like kinases, which play roles in regulating growth, development, and immunity. This largest family of receptor kinases in plants can be divided into subfamilies based on conservation of the kinase domain, which demonstrates that shared evolutionary history often indicates shared molecular function. Here we investigate the evolutionary history of this family across the evolution of 112 plant species. We identify lineage-specific expansions of the malectin-domain containing subfamily LRR subfamily I primarily in the Brassicales and bryophytes. Most other plant lineages instead show a large expansion in LRR subfamily XII, which in Arabidopsis is known to contain key receptors in pathogen perception. This striking asymmetric expansion may reveal a dichotomy in the evolutionary history and adaptation strategies employed by plants. A greater understanding of the evolutionary pressures and adaptation strategies acting on members of this receptor family offers a way to improve functional predictions for orphan receptors and simplify identification of novel stress related receptors.
Transcriptomic Effects of Low-Dose Inorganic Arsenic Exposure on Murine Bone Marrow-Derived Macrophages
Authors: Illingworth Emily J. , Maertens Alexandra , Sillé Fenna C.M.
Published: July 2023

Both tissue-resident macrophages and monocytes recruited from the bone marrow that transform into tissue-resident cells play critical roles in mediating homeostasis as well as in the pathology of inflammatory diseases. Inorganic arsenic (iAs) is the most common drinking water contaminant worldwide and represents a major public health concern. Several diseases that macrophages have implicated involvement in are caused by iAs exposure, including cardiovascular disease, cancer, and increased risk of infectious disease. Therefore, understanding the effects of iAs exposure on macrophages can help us better grasp the full range of arsenic immunotoxicity and better design therapeutic targets for iAs-induced diseases particularly in exposed populations. In this study, we analyzed the transcriptome of low dose iAs-exposed male and female murine bone marrow-derived macrophages (BMDMs) with either M0, M1, or M2 stimulation. We identified differentially expressed genes by iAs in a sex- and stimulation-dependent manner and used bioinformatics tools to predict protein-protein interactions, transcriptional regulatory networks, and associated biological processes. Overall, our data suggest that M1-stimulated, especially female-derived, BMDMs are most susceptible to iAs exposure. Most notably, we observed significant downregulation of major proinflammatory transcription factors, like IRF8, and its downstream targets, as well as genes encoding proteins involved in pattern recognition and antigen presentation, such as TLR7, TLR8, and H2-D1, potentially providing causal insight regarding arsenic’s role in perturbing immune responses to infectious diseases. We also observed significant downregulation of genes involved in processes crucial to coordinating a proinflammatory response including leukocyte migration, differentiation, and cytokine and chemokine production and response. Finally, we discovered that 24 X-linked genes were dysregulated in iAs-exposed female stimulation groups compared to only 3 across the iAs-exposed male stimulation groups. These findings elucidate the potential mechanisms underlying the sex-differential iAs-associated immune-related disease risk.
An allelic series at theEDNRB2locus controls diverse piebalding patterns in the domestic pigeon
Authors: Maclary Emily T. , Wauer Ryan , Phillips Bridget , Brown Audrey , Boer Elena F. , Samani Atoosa M. , Shapiro Michael D.
Published: July 2023

Variation in pigment patterns within and among vertebrate species reflects underlying changes in cell migration and function that can impact health, reproductive success, and survival. The domestic pigeon (Columba livia) is an exceptional model for understanding the genetic changes that give rise to diverse pigment patterns, as selective breeding has given rise to hundreds of breeds with extensive variation in plumage color and pattern. Here, we map the genetic architecture of a suite of pigmentation phenotypes known as piebalding. Piebalding is characterized by patches of pigmented and non-pigmented feathers, and these plumage patterns are often breed-specific and stable across generations. Using a combination of quantitative trait locus mapping in F2laboratory crosses and genome-wide association analysis, we identify a locus associated with piebalding across many pigeon breeds. This shared locus harbors a candidate gene,EDNRB2, that is a known regulator of pigment cell migration, proliferation, and survival. We discover multiple distinct haplotypes at theEDNRB2locus in piebald pigeons, which include a mix of protein-coding, noncoding, and structural variants that are associated with depigmentation in specific plumage regions. These results identify a role forEDNRB2in pigment patterning in the domestic pigeon, and highlight how repeated selection at a single locus can generate a diverse array of stable and heritable pigment patterns.

AUTHOR SUMMARY

Both wild and domestic birds show striking variation in pigment patterning, and these pigment patterns can play critical roles in mate choice, communication, and camouflage. Despite the importance of pigment patterning for survival and reproductive success, the mechanisms that control pigment patterning remain incompletely understood. In domestic pigeons, artificial selection has given rise to a wide array of pigmentation patterns within a single species, including a suite of phenotypes called “piebalding” that is characterized by regional loss of feather pigment. Here, we took advantage of the wide array of distinct piebalding phenotypes in domestic pigeons to map the genetic basis of region-specific loss of plumage pigment. Rather than focusing on a single piebalding pattern, we sought to broadly understand genetic control of regional pigment loss by examining several breeds with different piebalding patterns. We compared the genomes of piebald and non-piebald pigeons using both genetic crosses and genome-wide association studies, and identified several genetic changes affecting the endothelin receptor geneEDNRB2. Our findings highlight how independent mutations at a single locus can drive diversification of plumage pigment patterning within a species.
DNA methylation insulates genic regions from CTCF loops near nuclear speckles
Authors: Roseman Shelby A. , Siegenfeld Allison P. , Lee Ceejay , Lue Nicholas Z. , Waterbury Amanda L. , Liau Brian B.
Published: July 2023

The insulator protein CTCF is essential for mediating chromatin loops and regulating gene expression. While it is established that DNA methylation hinders CTCF binding, the impacts of this methylation-sensitive CTCF binding on chromatin architecture and transcription are poorly defined. Here, we used a selective DNMT1 inhibitor to investigate the characteristics and functions of ‘reactivated’ CTCF peaks resulting from global DNA demethylation. We found that reactivated peaks preferentially form chromatin loops on gene bodies and interact with highly-looping partner peaks located in regions of active chromatin. Notably, both reactivated CTCF peaks and their highly-looping partners are enriched near nuclear speckles, condensate bodies implicated in transcription and splicing. Utilizing targeted protein degradation, we specifically depleted CTCF and nuclear speckles to elucidate their functional interplay. By degrading CTCF upon DNMT1 inhibition, we revealed that CTCF is important for DNMT1 inhibitor-dependent interactions between chromatin and speckle proteins. Moreover, we found that CTCF promotes the activation of genes near speckles upon DNMT1 inhibition. Conversely, acute depletion of nuclear speckles revealed that they influence transcription but do not maintain CTCF binding or looping. Collectively, our study suggests a model wherein DNA methylation prevents spurious CTCF occupancy and interactions with regulatory elements near nuclear speckles, yet CTCF looping is robust towards the loss of speckles.
Evolution of the enhancer-rich regulatory region of thePOU1F1gene
Authors: Wallis Michael , Xu Qianlan , Krawczyk Michal , Skowronska-Krawczyk Dorota
Published: July 2023

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression ofPOU1F1is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5’ to 3’). We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting that conversion of alternative promoter to enhancer could be one evolutionary mechanism for enhancer birth. Our work shows that regulation ofPOU1F1changed markedly during the course of vertebrate evolution, use of a small number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of enhancers in mammals.
Teacher-student collaborated multiple instance learning for pan-cancer PDL1 expression prediction from histopathology slides
Authors: Jin Darui , Liang Shangying , Shmatko Artem , Arnold Alexander , Horst David , Grünewald Thomas G. P. , Gerstung Moritz , Bai Xiangzhi
Published: July 2023

Programmed cell death ligand 1 (PDL1), as an important biomarker, is quantified by immunohistochemistry with few established histopathological patterns. Deep learning aids in histopathological assessment, yet heterogeneity and lacking spatially resolved annotations challenge precise analysis. Here, we present a weakly supervised learning approach using bulk RNA sequencing for PDL1 expression prediction from hematoxylin and eosin (H&E) slides. Our methods, MILTS, extends multiple instance learning paradigm with the teacher-student framework, which assigns dynamic pseudo-labels for intra-slide heterogeneity and retrieves unlabeled instances using temporal ensemble model distillation. The approach, evaluated on 12,299 slides across 20 solid tumor types, achieves a weighted average AUC of 0.83 on fresh-frozen and 0.74 on formalin-fixed specimens for 9 tumors with PDL1 as an established biomarker. MILTS predicts PDL1 expression patterns, validated by immunohistochemistry on 20 slides, offering insights into histologies relevant to PDL1. This demonstrates the potential of deep learning in identifying diverse histological patterns for molecular changes from H&E images.