bluuebunny/crossref_metadata_2025 huggingface dataset:
The plasmid-borne hipBA operon of Klebsiella michiganensis encodes a potent plasmid stabilization system
Authors: Shutt-McCabe Jordan , Shaik Karimunnisa Begum , Hoyles Lesley , McVicker Gareth
Published: September 2024

Aims

Klebsiella michiganensis is a medically important bacterium that has been subject to relatively little attention in the literature. Interrogation of sequence data from K. michiganensis strains in our collection has revealed the presence of multiple large plasmids encoding type II toxin–antitoxin (TA) systems. Such TA systems are responsible for mediating a range of phenotypes, including plasmid stability (‘addiction’) and antibiotic persistence. In this work, we characterize the hipBA TA locus found within the Klebsiella oxytoca species complex (KoSC).

Methods and results

The HipBA TA system is encoded on a plasmid carried by K. michiganensis PS_Koxy4, isolated from an infection outbreak. Employing viability and plasmid stability assays, we demonstrate that PS_Koxy4 HipA is a potent antibacterial toxin and that HipBA is a functional TA module contributing substantially to plasmid maintenance. Further, we provide in silico data comparing HipBA modules across the entire KoSC.

Conclusions

We provide the first evidence of the role of a plasmid-encoded HipBA system in stability of mobile genetic elements and analyse the presence of HipBA across the KoSC. These results expand our knowledge of both a common enterobacterial TA system and a highly medically relevant group of bacteria.
Evaluation of mixed-mode stress intensity factors for anisotropic elastic solids with interface singularity
Authors: Zhang Wan-Ting , Hwu Chyanbin , Lin Tsung-Chi
Published: September 2024

When a structure is composed of many different parts and each part is made of different materials, it is very possible that many interface corners appear in several local fields of the structure. Due to the mismatch of elastic properties, stress singularity may occur at the interface corners. The singular orders of stresses and their associated stress intensity factors (SIFs) are two important parameters for understanding failure initiation. Since the singular orders of general interface corners may be real or complex, distinct or repeated, several different definitions of SIFs have been proposed for different types of interfaces. In order to build a direct connection among all general interface corners (including cracks and interface cracks), a unified definition of SIFs was proposed in our previous studies. To calculate the uni-defined SIF accurately and efficiently, a special boundary element using the fundamental solution satisfying interface continuity conditions is established. The singular integrals involved in the associated boundary integral equations are solved by the setting of proper rigid body motions and the approximation through interpolation of nearby non-singular solutions. To avoid the unstable and inaccurate region near the corners, in this study we employ the path-independent H-integral, and derive the relations between H-integral and SIFs. Moreover, the auxiliary solutions of displacements and tractions required in the H-integral are provided for different integral paths. Several numerical examples, such as interface cracks, interface corners, single lap joints and delamination, are presented to demonstrate the methodology employed in this paper.
Possible anticorrelations between pulsation amplitudes and the disc growth of Be stars in giant-outbursting Be X-ray binaries
Authors: Niwano Masafumi , Fausnaugh Michael M , Lau Ryan M , De Kishalay , Soria Roberto , Ricker George R , Vanderspek Roland , Ashley Michael C B , Earley Nicholas , Hankins Matthew J , Kasliwal Mansi M , Moore Anna M , Soon Jamie , Travouillon Tony , Sasada Mahito , Takahashi Ichiro , Yatsu Yoichi , Kawai Nobuyuki
Published: September 2024

The mechanism of X-ray outbursts in Be X-ray binaries remains a mystery, and understanding their circumstellar discs is crucial for a solution of the mass-transfer problem. In particular, it is important to identify the Be star activities (e.g. pulsations) that cause mass ejection and, hence, disc formation. Therefore, we investigated the relationship between optical flux oscillations and the infrared (IR) excess in a sample of five Be X-ray binaries. Applying the Lomb–Scargle technique to high-cadence optical light curves from the Transiting Exoplanet Survey Satellite (${\it TESS}$), we detected several significant oscillation modes in the 3–24 h period range for each source. We also measured the IR excess (a proxy for disc growth) of those five sources, using J-band light curves from Palomar Gattini-IR. In four of the five sources, we found anticorrelations between the IR excess and the amplitude of the main flux oscillation modes. This result is inconsistent with the conventional idea that non-radial pulsations drive mass ejections. We propose an alternative scenario where internal temperature variations in the Be star cause transitions between pulsation-active and mass-ejection-active states.
Analysis of an iterative reconstruction method in comparison of the standard reconstruction method
Authors: Chen Xinyi , Padmanabhan Nikhil
Published: September 2024

We present a detailed analysis of a new iterative density reconstruction algorithm. This algorithm uses a decreasing smoothing scale to better reconstruct the density field in Lagrangian space. We implement this algorithm to run on the quijote simulations, and extend it to (a) include a smoothing kernel that smoothly goes from anisotropic to isotropic, and (b) a variant that does not correct for redshift space distortions. We compare the performance of this algorithm with the standard reconstruction method. Our examinations of the methods include cross-correlation of the reconstructed density field with the linear density field, reconstructed two-point functions, and BAO parameter fitting. We also examine the impact of various parameters, such as smoothing scale, anisotropic smoothing, tracer type/bias, and the inclusion of second order perturbation theory. We find that the two reconstruction algorithms are comparable in most of the areas we examine. In particular, both algorithms give consistent fittings of BAO parameters. The fits are robust over a range of smoothing scales. We find the iterative algorithm is significantly better at removing redshift space distortions. The new algorithm will be a promising method to be employed in the ongoing and future large-scale structure surveys.
Thermophysical modelling of eclipse and occultation events in binary asteroid systems
Authors: Jackson Samuel L , Rozitis Benjamin
Published: September 2024

Binary systems comprise approximately 15 per cent of the near-Earth asteroid population, yet thermal-infrared (IR) data are often interpreted for these bodies as if they are single objects. Thermal-IR light curves of binary asteroids (3905) Doppler and (175706) 1996 FG3 are analysed using an adaptation of the Advanced Thermophysical Model, deriving new constraints on their thermal inertias as $\Gamma = 114 \pm 31\, \, \mathrm{J}\, \mathrm{m}^{-2}\, \mathrm{K}^{-1}\, \mathrm{s}^{-1/2}$ and $\Gamma = 142 \pm 6\, \, \mathrm{J}\, \mathrm{m}^{-2}\, \mathrm{K}^{-1}\, \mathrm{s}^{-1/2}$, respectively. We determine that this adapted model is suitable for binary systems where their primary rotation to secondary orbit period ratios can be approximately characterized by small integers. Objects with more complex orbital states require a model with alternative temperature convergence methodologies. Thermal inertia is shown to have a strong effect on binary thermophysical light-curve morphology, introducing significant modulations both inside and outside of mutual event times. The depths of eclipse events are shown to be suppressed at longer wavelengths due to the sensitivity to cooler parts of the surface, meanwhile surface roughness is shown to have little effect on the thermal light-curve morphology. A proof of concept model for the (65803) Didymos system is demonstrated, showing how such a binary model could be used to study the system during the European Space Agency’s Hera mission, and the applicability of this adapted model to NASA’s Lucy mission is also briefly discussed.
Kikuchi–Fujimoto disease and primary Sjögren’s syndrome coexisting: A case-based literature review
Authors: Cadório Maria João , Oliveira João , Gama João , Duarte Cátia
Published: September 2024

Kikuchi–Fujimoto Disease (KFD), also known as Kikuchi histiocytic necrotising lymphadenitis, is an extremely rare and benign condition that mostly affects young women. It is characterised by lymph node involvement with a predilection for the cervical region, commonly presenting with tender lymphadenopathy and a low-grade fever. The diagnosis requires excisional lymph node biopsy with immunohistochemical analysis. KFD is mostly self-limiting within a few weeks to months, with only some patients requiring symptomatic relief with non-steroidal anti-inflammatory drugs or corticosteroids, and a minority developing recurrent episodes of the disease. Importantly, it has been reported in association with systemic lupus erythematosus and, to a lesser extent, other immune-mediated inflammatory rheumatic diseases, such as Sjögren’s syndrome, whose clinical presentation itself may include lymphadenopathy. In this paper, we present an unusual case of a woman with primary Sjögren syndrome (pSS) and a past medical history relevant for lymphoma, sarcoidosis, and thymoma, who later developed KFD, a particularly challenging diagnosis in this setting. We then performed a literature review of the association between KFD and pSS, gathering a total of 13 patients, and focusing epidemiological, clinical, and laboratory data.
Canagliflozin and iron metabolism in the CREDENCE trial
Authors: Koshino Akihiko , Heerspink Hiddo J L , Jongs Niels , Badve Sunil V , Arnott Clare , Neal Bruce , Jardine Meg , Mahaffey Kenneth W , Pollock Carol , Perkovic Vlado , Hansen Michael K , Bakker Stephan J L , Wada Takashi , Neuen Brendon L
Published: September 2024

Background

Studies in patients with heart failure have indicated that sodium-glucose cotransporter 2 (SGLT2) inhibitors increase iron use and enhance erythropoiesis. In this post hoc analysis of the Canagliflozin and Renal Endpoints in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial, we evaluated the effects of canagliflozin on iron metabolism in patients with chronic kidney disease (CKD) and whether the effects of canagliflozin on hemoglobin and cardiorenal outcomes were modified by iron deficiency.

Methods

We measured serum iron, total iron binding capacity (TIBC), transferrin saturation (TSAT) and ferritin at baseline and 12 months. The effects of canagliflozin, relative to placebo, on iron markers were assessed with analysis of covariance. Interactions between baseline iron deficiency, defined as TSAT <20%, and the effects of canagliflozin on hemoglobin and cardiorenal outcomes were evaluated with mixed effect models and Cox regression models, respectively.

Results

Of 4401 participants randomized in CREDENCE, 2416 (54.9%) had iron markers measured at baseline, of whom 924 (38.2%) were iron deficient. Canagliflozin, compared with placebo, increased TIBC by 2.1% [95% confidence interval (CI) 0.4, 3.8; P = .014] and decreased ferritin by 11.5% (95% CI 7.1, 15.7; P < .001) with no clear effect on serum iron or TSAT. Canagliflozin increased hemoglobin over the trial duration by 7.3 g/L (95% CI 6.2, 8.5; P < .001) and 6.7 g/L (95% CI 5.2, 8.2; P < .001) in patients with and without iron deficiency, respectively (P for interaction = .38). The relative effect of canagliflozin on the primary outcome of doubling of serum creatinine, kidney failure or death due to cardiovascular disease or kidney failure (hazard ratio 0.70, 95% CI 0.56, 0.87) was consistent regardless of iron deficiency (P for interaction = .83), as were effects on other cardiovascular and mortality outcomes (all P for interactions ≥0.10).

Conclusion

Iron deficiency is highly prevalent in patients with type 2 diabetes and CKD. Canagliflozin increased TIBC and decreased ferritin in patients with type 2 diabetes and CKD, suggesting increased iron utilization, and improved hemoglobin levels and clinical outcomes regardless of iron deficiency.
Coronavirus pandemic derived demands and inclusive leadership on junior doctors’ well-being
Authors: Lin Y K , Lin B Y-J
Published: September 2024

Background

International studies have revealed that numerous challenges (e.g. job demands) and opportunities (e.g. job resources as protective factors) affect the well-being of healthcare workers.

Aims

Building on the job demands–resources theory, we explored how job-related demands and the role of inclusive leadership as a job resource influenced the well-being of junior doctors during the coronavirus disease 2019 (COVID-19) pandemic.

Methods

A cross-sectional web survey was conducted in July 2022 for 82 junior doctors who had experienced the 3-year COVID-19 pandemic. The survey captured their perceived physical, psychological and COVID-19-related job demands and the inclusive leadership behaviours exhibited by their clinical unit supervisors as job resources. Data were also collected on junior doctors’ experiences of burnout, compassion satisfaction and occupational commitment as variables of job outcomes. Hierarchical linear regression analysis was applied to explore the relationships between the variables.

Results

Physical and COVID-19-related job demands were associated with increased burnout among junior doctors. Inclusive leadership as a job resource was associated with junior doctors’ reduced burnout and increased compassion satisfaction and buffered the negative effects of psychological demands on their compassion satisfaction. Only COVID-19-related job demands were associated with junior doctors’ occupational commitment.

Conclusions

Our study highlights the critical effects of COVID-19-related job demands on junior doctors’ burnout and occupational commitment. Inclusive leadership behaviours exhibited by clinical unit supervisors are a valuable job resource, which can be incorporated as one of the competencies training for medical staff.
Denouncing the ‘One Voice’ Doctrine
Authors: Teo Marcus
Published: September 2024

The ‘one voice’ doctrine holds that the executive’s recognition of foreign states and governments is conclusive evidence of their status as such in English proceedings. However, the doctrine—properly understood as an irrebuttable presumption of status—is beset with theoretical and practical problems. Here, I argue that courts should abandon it, for three reasons: first, the doctrine is motivated by overbroad accounts of the executive’s foreign affairs prerogative; second, it suffers from inconsistencies on matters of scope, which its underlying justifications cannot resolve; and third, the doctrine creates conceptual incoherence, undermining the purpose of other doctrines which operate contingently upon it. In its place, courts should adopt an alternative rule, triggering a rebuttable presumption of status and attributing evidential weight to executive certificates, which avoids these problems while still serving the ‘one voice’ doctrine’s only legitimate purpose: helping courts reliably answer questions of status.
Light at the end of the tunnel: FRAP assays combined with super resolution microscopy confirm the presence of a tubular vacuole network in meristematic plant cells
Authors: Scheuring David , Minina Elena A , Krueger Falco , Lupanga Upendo , Krebs Melanie , Schumacher Karin
Published: September 2024

Plant vacuoles play key roles in cellular homeostasis, performing catabolic and storage functions, and regulating pH and ion balance. Despite their essential role, there is still no consensus on how vacuoles are established. A model proposing that the endoplasmic reticulum is the main contributor of membrane for growing vacuoles in meristematic cells has been challenged by a study proposing that plant vacuoles are formed de novo by homotypic fusion of multivesicular bodies (MVBs). Here, we use the Arabidopsis thaliana root as a model system to provide a systematic overview of successive vacuole biogenesis stages, starting from the youngest cells proximate to the quiescent center. We combine in vivo high- and super-resolution (STED) microscopy to demonstrate the presence of tubular and connected vacuolar structures in all meristematic cells. Using customized fluorescence recovery after photobleaching (FRAP) assays, we establish different modes of connectivity and demonstrate that thin, tubular vacuoles, as observed in cells near the quiescent center, form an interconnected network. Finally, we argue that a growing body of evidence indicates that vacuolar structures cannot originate from MVBs alone but receive membrane material from different sources simultaneously.
Analysing the scientific literature on policing approaches to disrupt child sexual exploitation
Authors: McVeigh Mary Jo , Heward-Belle Susan
Published: September 2024

Child sexual exploitation (CSE) is an insidious and far-reaching crime that harms children and young people in the short and long term and is a major area of concern for the Australasian police force. CSE frequently exists alongside other forms of criminal activity such as trafficking and gang-related activities. A major approach to combating CSE is the disruption of offenders’ activities. This article provides the result of a scoping review of the Australian and international literature to locate, analyse, and synthesize contemporary literature about disruptive policing tactics. The conceptual lens taken to review the literature was based on two research questions: (1) what police practices are utilized under the auspice of disruptive policing? and (2) how effective are disruptive policing tactics in preventing or protecting children and young people from CSE? Studies were included if they were in English, published between 2010 and 2023 in peer-reviewed journals or grey literature, and outlined disruptive police practices concerning CSE. The review found that although there is support for disruptive policing practices as a safeguarding tool to protect children and young people from perpetrators of CSE, there is a dearth of literature in this area. Australian police force youth liaison officers and Police Citizens Youth Clubs (PCYC) play a pivotal role in disrupting CSE. Effective disruptive activities are not the duty of the police force alone. Disrupting CSE requires a whole-of-government approach.
Research trends of computational toxicology: a bibliometric analysis
Authors: Yarmohammadi Fatemeh , Hayes A Wallace , Karimi Gholamreza
Published: September 2024

Background: Computational toxicology utilizes computer models and simulations to predict the toxicity of chemicals. Bibliometric studies evaluate the impact of scientific research in a specific field. Methods: A bibliometric analysis of the computational methods used in toxicity assessment was conducted on the Web of Science between 1977 and 2024 February 12. Results: Findings of this study showed that computational toxicology has evolved considerably over the years, moving towards more advanced computational methods, including machine learning, molecular docking, and deep learning. Artificial intelligence significantly enhances computational toxicology research by improving the accuracy and efficiency of toxicity predictions. Conclusion: Generally, the study highlighted a significant rise in research output in computational toxicology, with a growing interest in advanced methods and a notable focus on refining predictive models to optimize drug properties using tools like pkCSM for more precise predictions.
Single‐cell and spatial transcriptomics: Discovery of human placental development and disease
Authors: Tang Mi , Xiong Liling , Cai Jianghui , Fan Li , Huang Cheng , Zhang Shimao , Jin Ying , Luo Er‐dan , Xing ShaSha , Yang Xiao
Published: September 2024

The human placenta is a vital organ, encompassing many distinct cell types, that maintains the growth and development of the fetus and is essential for substance exchange, defense, synthesis, and immunity. Abnormalities in placental cells can lead to various pregnancy complications, but the mechanisms remain largely unclear. Single‐cell and spatial transcriptomics technologies have been developed in recent years to demonstrate placental cell heterogeneity and spatial molecular localization. Here, we review and summarize the current literature, demonstrating these technologies and showing the heterogeneity of various placenta cells and cell–cell communication of normal human placenta, as well as placenta‐related diseases, such as preeclampsia, gestational diabetes mellitus, advanced maternal age, recurrent pregnancy loss, and placenta accreta spectrum disorders. Meanwhile, current weaknesses and future direction were discussed.
Indole alleviates nonalcoholic fatty liver disease in an ACE2‐dependent manner
Authors: Gao Yuanyuan , Chen Qi , Yang Songtao , Cao Jie , Li Fangyu , Li Rui , Wu Zhuoying , Wang Ying , Yuan Li
Published: September 2024

Indole is a microbial metabolite produced by the gut microbiota through the degradation of dietary tryptophan, known for its well‐established anti‐inflammatory and antioxidant properties. In this study, we collected fecal samples from mice fed a high‐fat diet (HFD) and those on a standard diet (SD), then conducted 16S rRNA sequencing to analyze their gut microbiota. The analysis revealed distinct differences in the dominant bacterial species between the two groups, with a significant decrease in indole‐producing probiotics in the HFD mice compared to the SD group. Then we administered oral indole treatment to male C57BL/6J mice with HFD‐induced NAFLD and observed a significant improvement in hepatic steatosis and inflammation. Notably, indole alleviated the HFD‐induced decline in serum Angiotensin‐(1–7) [Ang‐(1–7)] levels and Angiotensin‐Converting Enzyme 2 (ACE2) expression. To further investigate the role of indole and ACE2 in NAFLD, we conducted experiments using ACE2 knockout (ACE2KO) mice that were also induced with HFD‐induced NAFLD and treated with indole. Interestingly, the protective effects of indole were compromised in the absence of ACE2. In HepG2 cells, indole similarly stimulated ACE2 expression and, in an ACE2‐dependent manner, reduced ROS generation, maintained mitochondrial membrane potential stability, and increased SIRT3 expression. In summary, our results highlight the formation of a biologically active gut‐liver axis between the gut microbiota and the liver through the tryptophan metabolite indole, which mitigates NAFLD in an ACE2‐dependent manner. Elevating dietary tryptophan and increasing indole levels may represent an effective approach for preventing and treating NAFLD.
Brown adipose tissue‐derived exosomes improve polycystic ovary syndrome in mice via STAT3/GPX4 signaling pathway
Authors: Fang Yu‐Qing , Zhang Han‐Ke , Wei Qiong‐Qiong , Li Yan‐Hui
Published: September 2024

Polycystic ovary syndrome (PCOS) is associated with impaired adipose tissue physiology. Elevated brown adipose tissue (BAT) mass or activity has shown potential in the treatment of PCOS. In this study, we aimed to investigate whether BAT‐derived exosomes (BAT‐Exos), as potential biomarkers of BAT activity, exert similar benefits as BAT in the treatment of PCOS. PCOS was induced in female C57BL/6J mice orally administered 1 mg/kg of letrozole for 21 days. Subsequently, the animals underwent transplantation with BAT or administered BAT‐Exos (200 μg) isolated from young healthy mice via the tail vein; healthy female mice were used as controls. The results indicate that BAT‐Exos treatment significantly reduced body weight and improved insulin resistance in PCOS mice. In addition, BAT‐Exos improved ovulation function by reversing the acyclicity of the estrous cycle, decreasing circulating luteinizing hormone and testosterone, recovering ovarian performance, and improving oocyte quality, leading to a higher pregnancy rate and litter size. Furthermore, western blotting revealed reduced expression of signal transducer and activator of transcription 3 (STAT3) and increased expression of glutathione peroxidase 4 (GPX4) in the ovaries of mice in the BAT‐Exos group. To further explore the role of the STAT3/GPX4 signaling pathway in PCOS mice, we treated the mice with an intraperitoneal injection of 5 mg/kg stattic, a STAT3 inhibitor. Consistent with BAT‐Exos treatment, the administration of stattic rescued letrozole‐induced PCOS phenotypes. These findings suggest that BAT‐Exos treatment might be a potential therapeutic strategy for PCOS and that the STAT3/GPX4 signaling pathway is a critical therapeutic target for PCOS.
Integrating various Experimental Information to Assist Protein Complex Structure Prediction by GRASP
Authors: Xie Yuhao , Zhang Chengwei , Li Shimian , Du Xinyu , Wang Min , Hu Yingtong , Liu Sirui , Gao Yi Qin
Published: September 2024

Protein complex structures are essential for understanding of biological activities and drug development. Improving complex structure prediction accuracy of AI models for cases such as antigen-antibody complexes is expected to further enhance their applicability. Meanwhile, a large variety of experimental methods are used to provide structural insights for protein complexes, with only sparse or approximate knowledge obtained. A general tool is needed to integrate AI models with limited experimental information for high-throughput and accurate protein complex structure prediction. To efficiently and flexibly incorporate the different forms of experimental information, we introduce here GRASP. GRASP outperforms existing tools in handling both simulated and real-world experimental restraints including those obtained from XL, CL, CSP, and DMS. As an example, GRASP excels in predicting antigen-antibody complex structures, even surpassing AF3 when utilizing experimental DMS and CL restraints. In addition to accelerating the restrained modeling process, its ability to integrate multiple forms of restraints makes it capable of integrative modeling. We also showcase its potential in modeling protein structural interactome in the near-cellular condition based on large-scalein vivoXL data for mitchondria.
Tricuspid valve leaflet remodeling in sheep with biventricular heart failure: A comparison between leaflets
Authors: Kostelnik Colton J. , Meador William D. , Lin Chien-Yu , Mathur Mrudang , Malinowski Marcin , Jazwiec Tomasz , Malinowska Zuzanna , Piekarska Magda L. , Gaweda Boguslaw , Timek Tomasz A. , Rausch Manuel K.
Published: September 2024

Tricuspid valve leaflets are dynamic tissues that can respond to altered biomechanical and hemodynamic loads. Each leaflet has unique structural and mechanical properties, leading to differential in vivo strains. We hypothesized that these intrinsic differences drive heterogeneous, disease-induced remodeling between the leaflets. Although we previously reported significant remodeling changes in the anterior leaflet, the responses among the other two leaflets have not been reported. Using a sheep model of biventricular heart failure, we compared the remodeling responses between all tricuspid leaflets. Our results show that the anterior leaflet underwent the most significant remodeling, while the septal and posterior leaflets exhibited similar but less pronounced changes. We found several between-leaflet differences in key structural and mechanical metrics that have been shown to contribute to valvular dysfunction. These findings underscore the need to consider leaflet-specific remodeling to fully understand tricuspid valve dysfunction and to develop targeted therapies for its treatment and more accurate computational models.

STATEMENT OF SIGNIFICANCE

Our study is significant as it advances our understanding of tricuspid valve remodeling by providing a comprehensive analysis of all three leaflets in a sheep model of biventricular heart failure. Unlike prior works that focused primarily on the anterior leaflet or generalized leaflet changes, we integrated morphological, histological, immunohistochemistry, biaxial mechanical testing, and two-photon microscopy to quantify differences between all three tricuspid valve leaflets (anterior, posterior, and septal) across multiple functional scales. This comprehensive approach highlights the unique remodeling response of each leaflet. Our findings offer critical insights for developing targeted therapeutic strategies and improving computational models of disease progression.

GRAPHICAL ABSTRACT
Modification and Analysis of Context-Specific Genome-Scale Metabolic Models: Methane-Utilizing Microbial Chassis as a Case Study
Authors: Kulyashov M.A. , Hamilton R. , Afshin Y. , Kolmykov S.K. , Sokolova T.S. , Khlebodarova T.M. , Kalyuzhnaya M.G. , Akberdin I.R.
Published: September 2024

Context-specific genome-scale model (CS-GSM) reconstruction is becoming an efficient strategy for integrating and cross-comparing experimental multi-scale data to explore the relationship between cellular genotypes, facilitating fundamental or applied research discoveries. However, the application of CS modeling for non-conventional microbes is still challenging. Here, we present a GUI interface that integrates COBRApy, EscherPy, and RIPTiDe Python-based tools within the BioUML platform and streamlines the reconstruction and interrogation of the CS genome-scale metabolic frameworks via Jupyter Notebook. The approach was tested using -omics data collected forMethylotuvimicrobium alcaliphilum20ZR, a prominent microbial chassis for methane capturing and valorization. We optimized the previously reconstructed whole genome-scale metabolic network by adjusting the flux distribution using gene expression data. The outputs of the automatically reconstructed CS metabolic network were comparable to manually optimizediIA409 models for Ca-growth conditions. However, the CS model questions the reversibility of the phosphoketolase pathway and suggested higher flux via primary oxidation pathways. The model also highlighted unresolved carbon partitioning between assimilatory and catabolic pathways at the formaldehyde-formate node. Only a very few genes and only one enzyme with a predicted function in C1-metabolism, a homolog of the formaldehyde oxidation enzyme (fae1-2), showed a significant change in expression in La-growth conditions. The CS-GSM predictions agreed with the experimental measurements under the assumption that the Fae1-2 is a part of tetrahydrofolate-linked pathway. The cellular roles of the tungsten (W)-dependent formate dehydrogenase (fdhAB) andfae-homologues (fae1-2,andfae3) were investigated via mutagenesis. The phenotype of the fdhABmutant followed the model prediction. Furthermore, more significant reduction of the biomass yield was observed during growth in La-supplemented media, confirming a higher flux through formate.M. alcaliphilum20ZRmutants lackingfae1-2did not display any significant defects in methane or methanol-dependent growth. However, contrary tofae,thefae1-2homolog failed to restore the formaldehyde activating enzyme function in complementation tests.

Overall, the presented data suggest that the developed computational workflow supports the reconstruction and validation of CS-GSM networks of non-model microbes.
Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity
Authors: Blazanin Nicholas , Liang Xiaobing , Mahmud Iqbal , Kim Eiru , Martinez Sara , Tan Lin , Chan Waikin , Anvar Nazanin Esmaeili , Ha Min Jin , Qudratullah Md , Minelli Rosalba , Peoples Michael , Lorenzi Philip , Hart Traver , Lissanu Yonathan
Published: September 2024

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex includingSMARCA4andARID1Ain non-small cell lung cancer. Previously, we and others have identified thatSMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.
Long-term dietary exposure to a mixture of phthalates enhances estrogen and beta-catenin signaling pathways, leading to endometrial hyperplasia in mice
Authors: Shukla Ritwik , Kannan Athilakshmi , Laws Mary J. , Johnson Amy Wagoner , Flaws Jodi A. , Bagchi Milan K. , Bagchi Indrani C.
Published: September 2024

Phthalates, synthetic chemicals widely utilized as plasticizers and stabilizers in various consumer products, present a significant concern due to their persistent presence in daily human life. While past research predominantly focused on individual phthalates, real-life human exposure typically encompasses complex mixtures of these compounds. The cumulative effects of prolonged exposure to phthalate mixtures on uterine health remain poorly understood. To address this knowledge gap, we conducted studies utilizing adult female mice exposed to a phthalate mixture for 6 and 12 months through ad libitum chow consumption. We previously reported that continuous exposure to this phthalate mixture for 6 months led to uterine fibrosis. In this study, we show that the exposure, when continued beyond 6 months to 1 year, caused fibrotic uteri to display hyperplasia with a significant increase in gland to stroma ratio. Endometrial hyperplasia is commonly caused by unopposed estrogen action, which promotes increased expression of pro-inflammatory cytokines and chemokines and proliferation of the endometrial epithelial cells. Indeed, RNA sequencing analysis revealed a marked upregulation of several estrogen-regulated genes, Wnt ligands that are involved in oncogenic pathways, as well as chemokines, in phthalate-exposed uterine tissues. Consequently, the exposed uteri exhibited increased proliferation of endometrial epithelial cells, and a heightened inflammatory response indicated by extensive homing of macrophages. Further studies revealed a marked enhancement of the Wnt/β-Catenin signaling pathway, potentially contributing to the development of endometrial hyperplasia. Collectively, this study underscores the significance of understanding the exposure to environmental factors in the pathogenesis of endometrial disorders.
TIRTL-seq: Deep, quantitative, and affordable paired TCR repertoire sequencing
Authors: Pogorelyy Mikhail V. , Kirk Allison M. , Adhikari Samir , Minervina Anastasia A. , Sundararaman Balaji , Vegesana Kasi , Brice David C. , Scott Zachary B. ,   SJTRC Study Team, Thomas Paul G.
Published: September 2024

ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies.

Here, we presentTIRTL-seq (Throughput-IntensiveRapidTCRLibrarysequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well.

The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired ɑβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ∼$200. We benchmarked TIRTL-seq against state-of-the-art 5’RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established.

The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.
Fibroblasts Regulate the Transformation Potential of Human Papillomavirus-positive Keratinocytes
Authors: James Claire D. , Lewis Rachel L. , Witt Austin J. , Carter Christiane , Rais Nabiha M. , Wang Xu , Bristol Molly L.
Published: September 2024

Persistent human papillomavirus (HPV) infection is necessary but insufficient for viral oncogenesis. Additional contributing co-factors, such as immune evasion and viral integration have been implicated in HPV-induced cancer progression. It is widely accepted that HPV+ keratinocytes require co-culture with fibroblasts to maintain viral episome expression, yet the exact mechanisms for this have yet to be elucidated. Here we present comprehensive RNA sequencing and proteomic analysis demonstrating that fibroblasts not only support the viral life cycle, but reduce HPV+ keratinocyte transformation. Our co-culture models offer novel insights into HPV-related transformation mechanisms.

Highlights

Fibroblasts support HPV RNA expression and episomal maintenance in HPV+ keratinocytes

Fibroblasts reduce EMT related expression in HPV+ keratinocytes

Fibroblasts promote EMT related expression in E6E7+ keratinocytes
Prediction and functional interpretation of inter-chromosomal genome architecture from DNA sequence with TwinC
Authors: Jha Anupama , Hristov Borislav , Wang Xiao , Wang Sheng , Greenleaf William J. , Kundaje Anshul , Aiden Erez Lieberman , Bertero Alessandro , Noble William Stafford
Published: September 2024

Three-dimensional nuclear DNA architecture comprises well-studied intra-chromosomal (cis) folding and less characterized inter-chromosomal (trans) interfaces. Current predictive models of 3D genome folding can effectively infer pairwisecis-chromatin interactions from the primary DNA sequence but generally ignoretranscontacts. There is an unmet need for robust models oftrans-genome organization that provide insights into their underlying principles and functional relevance. We present TwinC, an interpretable convolutional neural network model that reliably predictstranscontacts measurable through genome-wide chromatin conformation capture (Hi-C). TwinC uses a paired sequence design from replicate Hi-C experiments to learn single base pair relevance intransinteractions across two stretches of DNA. The method achieves high predictive accuracy (AUROC=0.80) on a cross-chromosomal test set from Hi-C experiments in heart tissue. Mechanistically, the neural network learns the importance of compartments, chromatin accessibility, clustered transcription factor binding and G-quadruplexes in formingtranscontacts. In summary, TwinC models and interpretstransgenome architecture, shedding light on this poorly understood aspect of gene regulation.
High quality Bathyarchaeia MAGs from lignocellulose-impacted environments elucidate metabolism and evolutionary mechanisms.
Authors: Nesbo camilla Lothe , Kublanov Ilya , Yang Minqing , Sharan Anupama Achal , Meyer Torsten , Edwards Elizabeth Anne
Published: September 2024

The archaeal class Bathyarchaeia are widely and abundantly distributed in anoxic habitats. Metagenomic studies have suggested that they are mixotrophic, capable of CO2 fixation and heterotrophic growth, and involved in acetogenesis and lignin degradation. We analysed 35 Bathyarchaeia metagenome assembled genomes (MAGs), including the first complete circularized MAG (cMAG) of the Bathy-6 subgroup, from the metagenomes of three full scale pulp and paper mill anaerobic digesters and three laboratory methanogenic enrichment cultures maintained on pre-treated poplar. Thirty-three MAGs belong to the Bathy-6 lineage while two are from the Bathy-8 lineage. In our previous analysis of the microbial community in the pulp mill digesters, Bathyarchaeia were abundant and positively correlated to hydrogenotrophic and methylotrophic methanogenesis. Several factors likely contribute to the success of the Bathy-6 lineage compared to Bathy-8 in the reactors. The Bathy-6 genomes are larger than those of Bathy-8 and have more genes involved in lignocellulose degradation including carbohydrate-active enzymes (CAZymes) not present in the Bathy-8. Bathy-6 also share the Bathyarchaeal O-demethylase-system recently identified in Bathy-8. All the Bathy-6 MAGs had numerous membrane-associated pyrroloquinoline quinone-domain (PQQ) proteins that we suggest are involved in lignin modification or degradation, together with Radical-SAM and Rieske domain proteins, and AA2, AA3, and AA6- family oxidoreductases. We also identified a complete B12 synthesis pathway and a complete nitrogenase gene locus. Finally, comparative genomic analyses revealed that Bathyarchaeia genomes are dynamic and have interacted with other organisms in their environments through gene transfer to expand their gene repertoire.
A Kinetic Scout Approach Accelerates Targeted Protein Degrader Development
Authors: Fan Angela T. , Gadbois Gillian E. , Huang Hai-Tsang , Jiang Jiewei , Sigua Logan H. , Smith Emily R. , Wu Sitong , Dunne-Dombrink Kara , Goyal Pavitra , Tao Andrew J. , Sellers William , Fischer Eric S. , Donovan Katherine A. , Ferguson Fleur M.
Published: September 2024

Bifunctional molecules such as targeted protein degraders induce proximity to promote gain-of-function pharmacology. These powerful approaches have gained broad traction across academia and the pharmaceutical industry, leading to an intensive focus on strategies that can accelerate their identification and optimization. We and others have previously used chemical proteomics to map degradable target space, and these datasets have been used to develop and train multiparameter models to extend degradability predictions across the proteome. In this study, we now turn our attention to develop generalizable chemistry strategies to accelerate the development of new bifunctional degraders. We implement lysine-targeted reversible-covalent chemistry to rationally tune the binding kinetics at the protein-of-interest across a set of 25 targets. We define an unbiased workflow consisting of global proteomics analysis, IP/MS of ternary complexes and the E-STUB assay, to mechanistically characterize the effects of ligand residence time on targeted protein degradation and formulate hypotheses about the rate-limiting step of degradation for each target. Our key finding is that target residence time is a major determinant of degrader activity, and this can be rapidly and rationally tuned through the synthesis of a minimal number of analogues to accelerate early degrader discovery and optimization efforts.
Clonal dynamics and somatic evolution of haematopoiesis in mouse
Authors: Kapadia Chiraag D. , Williams Nicholas , Dawson Kevin J. , Watson Caroline , Yousefzadeh Matthew J. , Le Duy , Nyamondo Kudzai , Cagan Alex , Waldvogel Sarah , De La Fuente Josephine , Leongamornlert Daniel , Mitchell Emily , Florez Marcus A. , Aguilar Rogelio , Martell Alejandra , Guzman Anna , Harrison David , Niedernhofer Laura J. , King Katherine Y. , Campbell Peter J. , Blundell Jamie , Goodell Margaret A. , Nangalia Jyoti
Published: September 2024

Haematopoietic stem cells maintain blood production throughout life. While extensively characterised using the laboratory mouse, little is known about how the population is sustained and evolves with age. We isolated stem cells and progenitors from young and old mice, identifying 221,890 somatic mutations genome-wide in 1845 single cell-derived colonies, and used phylogenetic analysis to infer the ontogeny and population dynamics of the stem cell pool. Mouse stem cells and progenitors accrue ∼45 somatic mutations per year, a rate only about 2-fold greater than human progenitors despite the vastly different organismal sizes and lifespans. Phylogenetic patterns reveal that stem and multipotent progenitor cell pools are both established during embryogenesis, after which they independently self-renew in parallel over life. The stem cell pool grows steadily over the mouse lifespan to approximately 70,000 cells, self-renewing about every six weeks. Aged mice did not display the profound loss of stem cell clonal diversity characteristic of human haematopoietic ageing. However, targeted sequencing revealed small, expanded clones in the context of murine ageing, which were larger and more numerous following haematological perturbations and exhibited a selection landscape similar to humans. Our data illustrate both conserved features of population dynamics of blood and distinct patterns of age-associated somatic evolution in the short-lived mouse.