bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique proteome signatures in ICU patients with COVID-19 and delirium: an observational study
Authors: Edel Andreas , Sreekanth Jayanth , Kurth Florian , Ralser Markus , Demichev Vadim , Mülleder Michael , Blanc Eric , Spies Claudia
Published: December 2024

Background

Delirium is common in COVID-19 intensive care unit (ICU) patients. Biomarkers for prediction, detection, and monitoring are missing. Unbiased omics analyses are warranted to gain a systems biology view on pathophysiology.

Methods

This prospective observational satellite study aims to investigate the proteome signatures of COVID-19 ICU patients, comparing those with delirium to those without. This study was conducted in ICUs of a university hospital between March 2020 and September 2021. ICU patients of legal age with a positive SARS-CoV-2 test were screened daily for oversedation and delirium. Blood samples were taken thrice a week. 457 samples were analyzed using data-independent acquisition mass spectrometry to determine protein levels. A mixed-effects logistic regression model was developed to identify proteins significantly influenced by delirium, accounting for sex and age as confounders. This model also aimed to determine proteins that were either up- or downregulated in association with delirium. Additionally, an enrichment analysis was conducted to examine the biological pathways linked to these delirium-associated proteins.

Results

Out of 360 ICU patients, 69 were analyzed for protein profiling. Out of these 69 patients, 42 patients (60.9%) had delirium on ICU admission, and 27 (39.1%) did not. Based on the multivariate model, the analysis of 204 proteins unfolded 125 (61.3%) to be differentially expressed. In total, 80.8% (n=101) of these 125 proteins were associated with delirium. Of these, 10 proteins were uniquely associated with delirium and were not significant in the multivariate model (SERPING1, SERPINA7, HP, TGFBI, CD5L, IGHV3-7, IGHV1-46, IGHV3-15, IGHV3-23, and “IGHV4-34;IGHV4-38-2”). In the univariate model for delirium, six out of 111 significant proteins showed increased expression with a log2FC > 0.5: PIGR, MST1, LBP, CRP, SAA1, and “SAA1;SAA2”; while three showed decreased expression with a log2FC < - 0.5: HP, PPBP, and “HP;HPR”. The enrichment analysis of delirium-influenced proteins revealed three significant pathways: “Network map of SARS-CoV-2 signaling” (M42569/WP5115), “Acute inflammatory response” (M10617), and “Regulation of defense response” (M15277).

Conclusion

We identified a unique proteomic signature in COVID-19 ICU patients with delirium, including up- and downregulated proteins. These findings may provide potential biomarker candidates for the assessment of delirium risk and its underlying causes. These findings could be a further step towards the development of personalized, causative treatments for delirium and its monitoring in the ICU.

Trial registration

The study was retrospectively registered in the German Clinical Trials Register on May 13, 2020 (DRKS00021688).
Hearing in Two Closely RelatedPeromyscusSpecies (Peromyscus maniculatusandP. leucopus)
Authors: Joseph Luberson , New Emily Margaret , Joseph Desi Marie , Woodley Tamara Chenell , Franco Vanessa Yamileth , Li Ben-Zheng , Wogan Guinevere OU , McCullagh Elizabeth A.
Published: December 2024

The genusPeromyscushas been extensively used as a model for ecological, behavioral, and evolutionary investigations. We used auditory brainstem responses (ABRs), craniofacial morphology, and pinna measurements to compare characteristics that impact hearing in two wild-caught species,P. leucopus P. maniculatus. We observed significant statistical differences in craniofacial and pinna attributes between species withP. leucopusoverall exhibiting larger features thanP. maniculatus. ABR recordings indicated that both species showed similar best frequency thresholds between 8-24 kHz. We found significant effects of intensity on amplitude ratio of wave I and IV forP. maniculatus, but notP. leucopusand effects of wave number on slope of the latency-intensity function with higher wave IV and shorter wave I slope of latency intensity function inP. leucopus. Finally, the data showed significant differences in latency shift of the DN1 component of the BIC in relation to ITD between species, while no significant differences were observed across relative DN1 amplitude. This study supports the used ofP. leucopusandP. maniculatusas future model species for auditory research.
Mitochondrial DNA mutations in human oocytes undergo frequency-dependent selection but do not increase with age
Authors: Arbeithuber Barbara , Anthony Kate , Higgins Bonnie , Oppelt Peter , Shebl Omar , Tiemann-Boege Irene , Chiaromonte Francesca , Ebner Thomas , Makova Kateryna D.
Published: December 2024

Mitochondria, cellular powerhouses, harbor DNA (mtDNA) inherited from the mothers. MtDNA mutations can cause diseases, yet whether they increase with age in human germline cells—oocytes—remains understudied. Here, using highly accurate duplex sequencing of full-length mtDNA, we detectedde novomutations in single oocytes, blood, and saliva in women between 20 and 42 years of age. We found that, with age, mutations increased in blood and saliva but not in oocytes. In oocytes, mutations with high allele frequencies (≥1%) were less prevalent in coding than non-coding regions, whereas mutations with low allele frequencies (<1%) were more uniformly distributed along mtDNA, suggesting frequency-dependent purifying selection. In somatic tissues, mutations caused elevated amino acid changes in protein-coding regions, suggesting positive or destructive selection. Thus, mtDNA in human oocytes is protected against accumulation of mutations having functional consequences and with aging. These findings are particularly timely as humans tend to reproduce later in life.

Graphical abstract
Deciphering the tumor-infiltrating CD73+ regulatory γδ T cell ecosystem associated with poor survival of patients with ovarian cancer
Authors: Chabab Ghita , Michaud Henri-Alexandre , Dejou Cécile , Chauvin Maeva , Chépeaux Laure-Agnès , Glasson Yaël , Boissière Florence , Lenain Marion , Dumé Anne-Sophie , Sarrant Pauline , Chemin Gabriel , Wajda Pauline , Dubois Bertrand , MacManus Anna , Colombo Pierre-Emmanuel , Fabbro Michel , Bonnefoy Nathalie , Lafont Virginie
Published: December 2024

The ability of tumor cells to overcome immune surveillance is an essential step in tumor development and progression. Among the immune cells playing a role in tumor control, γδ T cells contribute to the immune response against many tumor types through their direct cytotoxic activity against cancer cells and their capacity to regulate the functions of other immune cells. However, their presence in the tumor microenvironment is also associated with poor prognosis, suggesting that γδ T cells may also have pro-tumor activities. We previously described a regulatory γδ T-cell subset that expresses CD73 and produces IL-10, IL-8 and adenosine. Here, we report a higher CD73+ γδ T cell density in the tumor microenvironment of ovarian cancer samples from patients with short-term than long-term survival. Starting from this original observation, we investigated their neighborhood and described a specific ecosystem according to their pro-tumor functions.
Multi-omics analysis and genome-scale metabolic reconstruction of cattleBos taurusfor optimal production of cultured meat
Authors: Lee Junkyu , Kim Jaehyun , Bae Hyun Won , Kim Minji , Jung Byung Kwon , Kim Juyeon , Lee SungGun , Kim Hyun Uk
Published: December 2024

With the growing urgency of addressing climate change, cultured meat has gained significant attention as a sustainable alternative to conventional meat production.Bos taurus, a key cattle species, is considered as a potential source of cultured meat. However, much remains to be understood about the biology ofB. taurusmuscle cells. In this study, bovine satellite cells (BSCs) derived from the semimembranosus muscle of Korean Hanwoo cattle were subjected to multi-omics profiling and genome-scale metabolic reconstruction. First, differential gene expression and gene set enrichment analyses, based on RNA-seq data, identified key pathways associated with muscle cell proliferation (e.g., ‘Cell cycle’ and ‘RNA polymerase’) and differentiation (e.g., ‘Cytoskeleton in muscle cells’ and ‘Tryptophan metabolism’). Next, using the human1 GEM as a template, we constructed the firstB. taurus-specific genome-scale metabolic model (GEM), named BtaSBML2986, which comprises 2,986 genes, 13,278 reactions, and 8,652 metabolites. Muscle cells were cultured under six distinct conditions, and biomass predictions generated using BtaSBML2986 were validated against experimental growth rates. This integrated approach also provided insights into core pathways such as glycolysis and the TCA cycle. BtaSBML2986 represents a significant step forward in understandingB. taurusmuscle metabolism and will serve as a valuable tool for advancing cultured meat research and optimizing culture processes.
Deregulation of epigenetic factor, KDM6A, is involved during Candida albicans mediated delayed wound healing
Authors: Ghoshal Ankita , Shah Awantika , Balaji Kithiganahalli Narayanaswamy
Published: December 2024

Deregulation of wound healing process leads to chronic non-healing wounds. Opportunistic fungal pathogens likeCandida albicansis known to colonize open wounds leading to delayed, insufficient, or disorganized healing. In our study, we investigate the regulatory mechanisms orchestrating thisC. albicansmediated delay of wound healing and underscore a critical role for the transcription factor c-AMP Response Element Binding Protein (CREB) in regulating pro-inflammatory cytokines and genes implicated in wound healing. Parallelly, we found the epigenetic factor, KDM6A (Ubiquitously Transcribed tetratricopeptide repeat on chromosome X, also known as UTX) to be downregulated upon infection and modulating expression of metalloproteinases duringC. albicansgoverned wound healing impairment. Mechanistically, we observed enhanced recruitment of EZH2, a methyl transferase, over the promoters of KDM6A during infection, hence, governing its expression. Our findings, therefore, underscore a role of KDM6A and CREB inC. albicansmediated delayed wound healing.
Lipid Flipping, O-antigen Shielding, and Water Dynamics Revealed by 100-200 µs Coarse-Grained Simulations ofE. coliOuter Membrane Proteins
Authors: Kalenkiewicz Andrew , Elcock Adrian H.
Published: December 2024

Gram-negative (GN) bacterial infections are a major global health concern, with antibiotic-resistant strains representing a growing problem. The outer membrane (OM) of GN bacteria is the primary interface between the cell and its environment, and it plays a crucial role in regulating how GN bacteria interact with a host’s immune system and with antimicrobial drugs. Consequently, understanding the structural details of the GN OM is an essential step toward addressing the pressing threat of GN organisms. One way to gain insights into complex molecular structures like the GN OM is to use coarse-grained molecular dynamics (MD) simulations. In this work, we use theMartini3coarse-grained force field to model and simulate ten different OM proteins (OMPs) fromE. coliembedded in four different OM models that each contain lipopolysaccharides (LPS) with different O-antigen lengths. We extensively assess OMP-phospholipid interactions and demonstrate that TolC, in particular, shows a strong preference for binding cardiolipin. We also uncover a phenomenon that, to our knowledge, has not been previously described in the simulation literature—i.e., the ability of OMPs to serve as sites of phospholipid flipping from the OM inner leaflet to the outer leaflet. Finally, we show that longer LPS O-antigen chains occlude OMPs, and we provide a detailed characterization of water and ion movement throughout our simulations.
Endosome maturation is orchestrated by inside-out proton signaling through a Na+/H+ exchanger and pH-dependent Rab GTPase cycling
Authors: Lee YouJin , Ouyang Qing , Riaz Hasib Aamir , Ma Li , Fleishman Morgan , Schmidt Michael , Dupree Jeffrey L , Lambright David G , Morrow Eric M
Published: December 2024

Endosome maturation requires progressive lumen acidification. To what extent is lumen acidification sensed by cytosolic-side molecules that drive endosome maturation? We show here that "inside-out" proton signaling through the endosomal Na+/H+ Exchanger 6 (NHE6) activates the late endosome master regulator Rab7. The mechanism involves potent inactivation of the Rab7 GTPase-activating protein (GAP) TBC1D5 with decreasing pH. NHE6 interacts with TBC1D5 in a complex with Rab7. Neurons from NHE6-null mice or mice engineered with a selective defect in NHE6 proton efflux exhibit blocked endosome maturation and decreased active Rab7, consistent with an overactive Rab7 GAP. Finally, epistatic knock-down of TBC1D5, thereby reducing Rab7 GAP activity, in NHE6-null neurons rescues Rab7 GTPase cycling and endosome maturation. Importantly, NHE6 is mutated in Christianson Syndrome underscoring the significance of these mechanisms to neurodegeneration. We conclude that lumen acidification regulates pH-dependent Rab GTPase cycling to coordinate late endosome maturation by a process involving proton signaling.
Trophic level associated gut length divergence evolved under sexual conflict in Lake Malawi cichlids
Authors: Carmona Baez Aldo , Ciccotto Patrick J. , Moore Emily C. , Peterson Erin N. , Lamm Melissa S. , Roberts Natalie B. , Coyle Kaitlin P. , Barker M. Kaitlyn , Dickson Ethan , Cass Amanda N. , Pereira Guilherme S. , Zeng Zhao-Bang , Guerrero Rafael F. , Roberts Reade B.
Published: December 2024

Variation in gastrointestinal morphology is associated with dietary specialization across the animal kingdom. Gut length generally correlates with trophic level, and increased gut length in herbivores is a classic example of adaptation to cope with diets with lower nutrient content and a higher proportion of refractory material. However, the genetic basis of gut length variation remains largely unstudied, partly due to the inaccessibility and plasticity of the gut tissue, as well as the lack of dietary diversity within traditional model organisms relative to that observed among species belonging to different trophic levels. Here, we confirm the genetic basis of gut length variation among recently evolved Lake Malawi cichlid fish species with different dietary adaptations. We then produce interspecific, inter-trophic-level hybrids to map evolved differences in intestinal length in an F2 mapping cross betweenMetriaclima mbenjii, an omnivore with a relatively long gut, andAulonocara koningsi, a carnivore with a relatively short gut. We identify numerous candidate quantitative trait loci for evolved differences in intestinal length. These quantitative trait loci are predominantly sex-specific, supporting an evolutionary history of sexual conflicts for the gut. We also identify epistatic interactions potentially associated with canalization and the maintenance of cryptic variation in the cichlid adaptive radiation. Overall, our results suggest a complex, polygenic evolution of gut length variation associated with trophic level differences among cichlids, as well as conflicts and interactions that may be involved in evolutionary processes underlying other traits in cichlids.

This study examines the genetic basis of gut length variation in Lake Malawi cichlids, which exhibit different dietary adaptations. It highlights how cichlids recapitulate a broad taxonomic trend: gut length correlates with trophic level, with herbivores and omnivores having longer intestines than carnivores. By creating hybrids ofMetriaclima mbenjii(omnivore) andAulonocara koningsi(carnivore), we identify several quantitative trait loci and epistatic interactions underlying gut length differences. These genetic associations are predominantly sex-specific, suggesting historical sexual conflicts. The results indicate complex, polygenic evolution of gut morphology in these fish, and suggest evolutionary interactions and processes shaping dietary traits across species.
Cryo-EM of PMEL Amyloids Reveals Pathogenic Mechanism of Pigment Dispersion Syndrome
Authors: Yanagisawa Haruaki , Arai Harumi , Miyazawa Hideyuki , Kikkawa Masahide , Oda Toshiyuki
Published: December 2024

PMEL amyloids provide a vital scaffold for melanin deposition in melanosomes, playing a central role in pigmentation. Despite their importance, the high-resolution structure of PMEL amyloids has remained elusive. Here, we determined near-atomic resolution structures of wild-type PMEL amyloids using cryo-electron microscopy, revealing two distinct polymorphic forms with unique structural features. We further examined the pathogenic G175S mutation linked to pigment dispersion syndrome (PDS). Structural analysis showed that the G175S mutation introduces an additional hydrogen bond, stabilizing a novel fibril conformation. In vitro assays demonstrated a fourfold increase in polymerization efficiency for the G175S mutant compared to the wild-type. This enhanced polymerization correlated with a ∼70% increase in secreted amyloids in G175S-expressing cells without detectable changes in melanosome morphology or number. These findings suggest that the G175S mutation promotes amyloidogenesis within melanosomes, increasing amyloid load and contributing to PDS pathophysiology. This study provides insights into the molecular basis of PMEL amyloid formation in both physiological and pathological contexts, offering new perspectives on their structural diversity and dysregulation in pigmentation disorders.
Large MAF Transcription Factors Reawaken Evolutionarily Dormant Fast-Glycolytic Type IIb Myofibers in Human Skeletal Muscle
Authors: Sadaki Shunya , Tsuji Ryosuke , Hayashi Takuto , Watanabe Masato , Iwai Ryoto , Wenchao Gu , Semenova Ekaterina A. , Sultanov Rinat I. , Zhelankin Andrey V. , Generozov Edward V. , Ahmetov Ildus I. , Sakakibara Iori , Ojima Koichi , Sakurai Hidetoshi , Muratani Masafumi , Kudo Takashi , Takahashi Satoru , Fujita Ryo
Published: December 2024

Small mammals rely on type IIb myofibers, expressing the fastest myosin IIb (encoded byMYH4), for rapid muscle contraction. In contrast, larger mammals, including humans, show reduced or absentMYH4expression and type IIb myofibers, favoring slower-contracting myofibers. The evolutionary mechanisms underlying this shift remain unclear. Here, we identify large MAF transcription factors (MAFA, MAFB, MAF) as key regulators ofMYH4expression in large mammals, including human and bovine. Overexpression of large MAFs induces MYH4 expression and enhances glycolytic capacity in human myotubes, supported by RNA-seq and metabolic flux analyses. RNA-seq of human muscle biopsies reveals a positive correlation betweenMAFA,MAF, andMYH4expression, with these genes elevated in power-trained individuals. These findings reveal a conserved mechanism across mammals, showing that large MAFs can induce type IIb myofibers even in humans, with potential applications for enhancing athletic performance and addressing age-related muscle weakness associated with the loss of fast-twitch myofibers.
Distribution of Time Documented as Spent on Observation Unit Disposition Process
Authors: Edmond Farrah , Algarin Mojica Nicole , Horowitz Samantha , Ng Htet , Bacchus Matthew , Harharsh Tarek , Richman Mark
Published: December 2024

Introduction

Emergency Department (ED) Observation Units (OUs) are increasingly-common. Centers for Medicare and Medicaid Services (CMS) reimbursement policies incentivize documenting longer times spent on the disposition/discharge process (≥31 minutes). In addition, there is a well-recognized phenomenon of “terminal digit preference (ending times in 0 or 5). The Long Island Jewish Medical Center (LIJ) ED’s OU disposition process is performed by physician assistants (PAs). This study investigates the distribution of times documented by OU PAs as being spent on disposition and whether those times indicate terminal digit preference.

Methods

Documentation for discharge times of 102 patients dispositioned from the LIJ ED OU were captured, along with the PA involved. Students T-test was used to compare percentages. Statistical significance was set a priori at p <0.05.

Results

Seven PAs entered discharge times for 102 patients. No times were documented as <31 minutes. Time documented by PAs demonstrated significant clustering (ie, each PA was very consistent in the amount of time they documented). Nearly 50% of times were documented as “35 minutes.” Almost the entire remainder of times (46.1%) were documented as exactly 31 minutes or within 2 minutes of that (ie, 31, 32, or 33 minutes). Slightly over 50% of times documented ended in a “0” or a “5,” statistically-significantly greater than the expected 20% (p <0.0001).

Discussion

Documentation of most times being at or just slightly greater than 31 minutes suggests that documentation may reflect CMS’s reimbursement incentives. Over half of times documented ended in 0 or 5, indicating “terminal digit preference.” Further research is needed to confirm these findings and analyze the impact of CMS policy changes on in healthcare documentation.
Resolving inter- and intra-patient heterogeneity inNPM1-mutated AML at single-cell resolution
Authors: Karakaslar E Onur , Argiro Eva M , Struckman Nadine E , Shirali Ramin HZ , Severens Jeppe F , Honders M Willy , Kloet Susan L , Veelken Hendrik , Reinders Marcel JT , Griffioen Marieke , van den Akker Erik B
Published: December 2024

NPM1-mutated AML is one of the largest entities in international classification systems of myeloid neoplasms, which are based on integrating morphologic and clinical data with genomic data. Previous research, however, indicates that bulk transcriptomics-based subtyping may improve prognostication and therapy guidance. Here, we characterized the heterogeneity inNPM1-mutated AML by performing single-cell RNA-sequencing and spectral flow cytometry on 16 AML belonging to three distinct subtypes previously identified by bulk transcriptomics. Using single-cell expression profiling we generated a comprehensive atlas ofNPM1-mutated AML, collectively reconstituting complete myelopoiesis. The threeNPM1-mutated transcriptional subtypes showed consistent differences in the proportions of myeloid cell clusters with distinct patterns in lineage commitment and maturational arrest. In all samples, malignant cells were detected across different myeloid cell clusters, indicating thatNPM1-mutated AML are heavily skewed but not fully arrested in myelopoiesis. Same-sample multi-color spectral flow cytometry recapitulated these skewing patterns, indicating that the threeNPM1-mutated subtypes can be consistently identified across platforms. Moreover, our analyses highlighted differences in the abundance of rare hematopoietic stem cells suggesting that skewing occurs early in myelopoiesis. To conclude, by harnessing single-cell RNA-sequencing and spectral flow cytometry, we provide a detailed description of three distinct and reproducible patterns in lineage skewing inNPM1-mutated AML that may have potential relevance for prognosis and treatment of patients withNPM1-mutated AML.

KEY MESSAGES

NPM1-mutated AML shows strong intra- and interpatient heterogeneity with malignant cells skewed rather than fully arrested at different maturation stages in myelopoiesis.

Single-cell RNA sequencing and spectral flow cytometry revealed recurrent patterns in proportions of malignant myeloid cells with distinct patterns in lineage commitment and maturational arrest.

GRAPHICAL ABSTRACT
Investigating the pathogenicity of the recessiveHNF1Ap.A251T variant in monogenic diabetes using iPSC-derived beta-like cells
Authors: Cherkaoui Ines , Du Qian , Egli Dieter M. , Dion Camille , Leitch Harry G. , Sachedina Dilshad , Misra Shivani , Rutter Guy A.
Published: December 2024

Monogenic diabetes, formerly called Maturity-Onset Diabetes of the Young (MODY), involves single-gene mutations, typically with dominant inheritance, and has been associated with variants in 14 genes. Among these,HNF1Amutations are the most common, and their diagnosis allows the use of alternative therapies, including sulfonylureas. In an earlier study, we described a variant displaying recessive transmission, p.A251T (Misra, S et al,Diabetes Care, 2020). Initial functional studies revealed only a modest impact on protein function. We extend these earlierin vitrostudies to demonstrate that beta-like cells derived from pluripotent stem cells from variant carriers show impaired differentiation into insulin-positive cells, whereas differentiation into alpha cells is significantly enhanced. Additionally, mutant cells showed impaired glucose-stimulated insulin secretion but partially preserved responsiveness to treatment with sulfonylureas. Our study provides proof of principle for the utility of using patient-derived stem cells as a platform to assess the pathogenicity ofHNF1Avariants, and to explore potential treatment strategies.
Patient demographics, medical factors, treatment modalities and satisfaction at five traditional Chinese medicine practices: A cross-sectional study
Authors: Liu Yingchao , Lyu Xiaoying , Pradhan Saroj K. , Li Yiming , Liu Xingfang , Bauder Ralf , Heggli Tanja , Wang Xiaying , Furian Michael
Published: December 2024

1

BACKGROUND

Traditional Chinese Medicine (TCM) is increasingly integrated into healthcare and insurance systems, therefore, it is essential to understand its current status and patients’ perspectives.

METHODS

This cross-sectional study was conducted from January 1st to December 31st, 2023, across five TCM practices in Switzerland. All patients attending their sixth therapy session were invited to complete an electronically anonymized questionnaire covering patient demographics, treatment experiences, and satisfaction.

RESULTS

A total of 461 patients participated in the survey, with the majority being female (60.1%) and aged 50 years or older (57.4%). Among them, 54.0% reported multiple health conditions, with 32.9% having musculoskeletal disorders and 31.8% suffering from chronic pain as the main reasons for seeking therapy. Most patients received weekly TCM treatments (91.3%), with 50.7% also undergoing conventional therapies. Of the respondents, 50.0% reported full coverage for their TCM therapy costs. TCM was mainly accessed through personal recommendations (44.5%), and 92.2% experienced wait times under 10 minutes. Acupuncture was the predominant treatment (95.7%), with 35.8% receiving additional dietary advice. Overall satisfaction reached 96.5%, and 99.5% expressed intent to continue TCM. Full compared to no TCM expense coverage was positively associated with treatment satisfaction (odds ratio = 2.42, 95% CI [1.10 to 5.31], p = 0.028), while other medical factors showed no significant impact on satisfaction.

CONCLUSION

This study indicates that women, patients over 50 years, and those with multiple health conditions, especially musculoskeletal and pain conditions, are more likely to seek regular integrated TCM treatment. Patients reported high satisfaction with TCM, and treatment satisfaction was positively associated with full compared to no health insurance coverage. Future research should include more TCM practices and patients to further generalize these findings and precisely assess the impact of insurance coverage on TCM satisfaction to meet patient needs better.
Ancestral aryl sulfatases in the alkaline phosphatase family allow for greater increases in catalytic efficiency than extant variants
Authors: Eenink Bernard , Heberlein Magdalena , Holstein Josephin M. , Lange Andreas , Dilkaute Carina , Kaminski Tomasz S. , Bornberg-Bauer Erich , Jose Joachim , Hollfelder Florian , van Loo Bert
Published: December 2024

Characterizing the dynamics and functional shifts during protein evolution is essential, both for understanding protein evolution and for rationalizing efficient strategies for e.g. enzymes with desired and effective functions.

Most proteins organize in families, sets of divergent sequences which share a common ancestor and a have a similar structural fold.

We study Aryl sulfatases (ASs), a subfamily of the large and evolutionary old alkaline phosphatase superfamily. Together with their computationally reconstructed putative common ancestors, we expressed and enzymatically characterized these enzymes using micro-fluidics generated high-throughput libraries.

We compare the evolvability and robustness of ancestors and four extant ASs which all exhibit catalytic promiscuity towards a range of substrate classes.

Sixteen libraries with distinct mutational loads were expressed in individual cells inside micro-droplets.

We find that higher mutational loads yielded a longer tail of active variants but fewer total improved variants.

The effects of mutational load varied quantitatively between enzymes, meaning in some enzymes a higher, in others a lower mutational load was beneficial, highlighting the importance of testing various mutagenesis regimes.

Although ancestors did not display the highest proportion of active variants, they did exhibit the greatest activity increases.

We conclude that in ASs the ancestors are not necessarily more promiscuous but the fitness landscape around them is more shallow and thus more amenable to evolutionary optimization.
E-cadherin endocytosis promotes non-canonical EGFR:STAT signalling to induce cell death and inhibit heterochromatinisation
Authors: Ramirez-Moreno Miguel , Quinton Amy , Jacobsen Eleanor , Stempor Przemyslaw A. , Zeidler Martin P. , Bulgakova Natalia A.
Published: December 2024

Signalling molecules often contribute to several signalling pathways that produce distinct transcriptional outputs and cellular phenotypes. One of the major unanswered questions in cell biology is how multiple activities of signalling molecules are coordinated in space and time in vivo. Here, we focus on the Signal Transducer and Activator of Transcription (STAT) protein as a paradigm of signalling molecules involved in several independent signalling pathways. In addition to the canonical pathways, whereby STAT is phosphorylated and activated by Janus Kinases, STAT is involved in at least two non-canonical pathways. In one pathway, STAT is activated by the Epidermal Growth Factor Receptor (EGFR) promoting apoptosis. In another, it binds the Heterochromatin Protein 1 (HP1) to enhance heterochromatin formation. UsingDrosophilawing discs as an epithelial system, we demonstrate that all three STAT activities coexist in this tissue. We provide evidence that while the canonical STAT signalling is dominant over non-canonical pathways, EGFR:STAT and HP1:STAT pathways compete for the availability of unphosphorylated STAT. We also describe the role of the cell-cell adhesion protein E-cadherin in EGFR:STAT signalling. Both EGFR and STAT colocalise with E-cadherin at cell-cell junctions and on intracellular vesicles. Elevated intracellular E-cadherin promotes EGFR:STAT pathway leading to apoptosis, whereas blocking E-cadherin endocytosis prevents apoptosis induction. Altogether, we propose that E-cadherin endocytosis controls the balance between two non-canonical STAT activities in a potential tumour-suppressive mechanism – junctional disassembly in dysregulated epithelial-to-mesenchymal transitions would shift this balance towards the EGFR:STAT signalling to promote apoptosis.
Oncogenic RAS Induces a Distinctive Form of Non-Canonical Autophagy Mediated by the P38-ULK1-PI4KB Axis
Authors: Wang Xiaojuan , Li Shulin , Lin Shiyin , Han Yaping , Zhan Tong , Huang Zhiying , Wang Juanjuan , Li Ying , Deng Haiteng , Zhang Min , Feng Du , Ge Liang
Published: December 2024

Cancer cells with RAS mutations exhibit enhanced autophagy, essential for their proliferation and survival, making it a potential target for therapeutic intervention. However, the regulatory differences between RAS-induced autophagy and physiological autophagy remain poorly understood, complicating the development of cancer-specific anti-autophagy treatments. In this study, we identified a form of non-canonical autophagy induced by oncogenic KRAS expression, termed RAS-induced non-canonical autophagy via ATG8ylation (RINCAA). RINCAA involves distinct autophagic factors compared to those in starvation-induced autophagy and incorporates non-autophagic components, resulting in the formation of non-canonical autophagosomes with multivesicular/multilaminar structures labeled by ATG8 family proteins (e.g., LC3 and GABARAP). We have designated these structures as RAS-induced multivesicular/multilaminar bodies of ATG8ylation (RIMMBA). A notable feature of RINCAA is the substitution of the class III PI3K in canonical autophagy for PI4KB. We identified a regulatory P38-ULK1-PI4KB-WIPI2 signaling cascade governing this process, where ULK1 phosphorylation at S317, S479, S556, and S758 activates PI4KB. This activation involves PI4KB phosphorylation at S256 and T263, initiating PI4P production, ATG8ylation, and non-canonical autophagy. Importantly, elevated PI4KB phosphorylation at S256 and T263 was observed in RAS-mutated cancer cells and colorectal cancer specimens. Inhibition of PI4KB S256 and T263 phosphorylation led to a reduction in RINCAA activity and tumor growth in both xenograft and KPC models of pancreatic cancer, suggesting that ULK1-mediated PI4KB-Peptide-1 phosphorylation could represent a promising therapeutic target for RAS-mutated cancers.
Depositing biological segmentation datasets FAIRly
Authors: Ho Elaine M. L. , Ladakis Dimitrios , Basham Mark , Darrow Michele C
Published: December 2024

Segmentation of biological images identifies regions of an image which correspond to specific features of interest, which can be analysed quantitatively to answer biological questions. This task has long been a barrier to conducting large-scale biological imaging studies as it is time- and labour-intensive. Modern artificial intelligence segmentation tools can automate this process, but require high quality segmentation data for training, which is challenging to acquire. Biological segmentation data has been produced for many years, but this data is not often reused to develop new tools as it is hard to find, access, and use. Recent disparate efforts (Iudin, et al., 2023; Xu, et al., 2021; Vogelstein, et al., 2018; Ermel, et al., 2024) have been made to facilitate deposition and re-use of these valuable datasets, but more work is needed to increase re-usability. In this work, we review the current state of publicly available annotation and segmentation datasets and make specific recommendations to increase re-usability following FAIR (findable, accessible, interoperable, re-usable) principles (Wilkinson, et al., 2016) for the future.
I/σIvs{Rmerg, Rmeas, Rpim, CC1/2} for Crystal Diffraction Data Quality Evaluation
Authors: Fu Zheng-Qing , Geisbrecht Brian V. , Bouyain Samuel , Dyda Fred , Chrzas John , Kandavelu Palani , Miller Darcie J. , Wang Bi-Cheng
Published: December 2024

X-ray crystal diffraction has provided atomic-level structural information on biological macromolecules. Data quality determines the reliability of structural models. In most cases, multiple data sets are available from different crystals and/or collected with different experimental settings. Reliable metrics are critical to rank and select the data set with the highest quality. Many measures have been created or modified for data quality evaluation. However, some are duplicate in functionality, and some are likely misused due to misunderstanding, which causes confusion or problems, especially at synchrotron beamlines where experiments proceed quickly. In this work, these measures are studied through both theoretical analysis and experimental data with various characteristics, which demonstrated that: 1). {Rmerg, Rmeas, Rpim, CC1/2} all measure the equivalence of reflections, and the low-shell values of these metrics can be used as reliable indicators for correctness (or trueness) of Laue symmetry; 2). High-shell I/σI is a reliable and better indicator to select resolution cutoff while the overall value measures the overall strength of the data.
Recognizing amino acid sidechains in a medium resolution cryo-electron density map
Authors: Mondal Dibyendu , Kumar Vipul , Satler Tadej , Ramachandran Rakesh , Saltzberg Daniel , Chemmama Ilan , Pilla Kala Bharath , Echeverria Ignacia , Webb Benjamin M. , Gupta Meghna , Verba Kliment A , Sali Andrej
Published: December 2024

Building an accurate atomic structure model of a protein into a cryo-electron microscopy (cryo-EM) map at worse than 3 angstrom resolution is difficult. To facilitate this task, we devised a method for assigning the amino acid residue sequence to the backbone fragments traced in an input cryo-EM map (EMSequenceFinder). EMSequenceFinder relies on a Bayesian scoring function for ranking 20 standard amino acid residue types at a given backbone position, based on the fit to a density map, map resolution, and secondary structure propensity. The fit to a density is quantified by a convolutional neural network that was trained on ~5.56 million amino acid residue densities extracted from cryo-EM maps at 3-10 angstrom resolution and corresponding atomic structure models deposited in the Electron Microscopy Data Bank (EMDB). We benchmarked EMSequenceFinder by predicting the sequences of 58,044 distinct ɑ-helix and β-strand fragments, given the fragment backbone coordinates fitted in their density maps. EMSequenceFinder identifies the correct sequence as the best-scoring sequence in 77.8% of these cases. We also assessed EMSequenceFinder on separate datasets of cryo-EM maps at resolutions from 4 to 6 angstrom. The accuracy of EMSequenceFinder (63.5%) was better than that of two tested state-of-the-art methods, including findMysequence (45%) and sequence_from_map in Phenix (12.9%). We further illustrate EMSequenceFinder by threading the SARS-CoV-2 NSP2 sequence into eight cryo-EM maps at resolutions from 3.7 to 7.0 angstrom. EMSequenceFinder is implemented in our open-source Integrative Modeling Platform (IMP) program. Thus, it is expected to be helpful for integrative structure modeling based on a cryo-EM map and other information, such as models of protein complex components and chemical crosslinks between them.
Comparison of infliximab versus cyclophosphamide on treatment of refractory uveitis of Behcet’s disease: pilot clinical study
Authors: Sadeghi Alireza , Karimimoghadam Arezoo , Davatchi Fereydoun , Shahram Farhad , Mazloomzadeh Saeideh , Behnamghader Azadeh , Jalili Nooshin , Biglari Sajjad , Jourahmad Zahra
Published: December 2024

Background

To evaluate the effect of infliximab (IFX), in comparison with cyclophosphamide (CP) on refractory uveitis of BD.

Methods

Subjects with uveitis and retinal vasculitis (RV) resistant to conventional treatment were included. Ten patients were divided in the IFX and CP groups. Patients in the IFX group received four courses of 3 mg/kg of IFX, intravenously. Patients in the CP group received four courses of pulse CP of 1000 mg, by the monthly interval. All patients received weekly 25 mg of methotrexate, (or 5 mg/kg of cyclosporine daily), and 3 mg/kg and 1 mg/kg of daily azathioprine and prednisolone, respectively. To evaluate the treatment effects, visual acuity (VA), anterior uveitis (AU), posterior uveitis (PU), and RV were assessed, at the baseline and four months after treatment.

Results

In ten patients included in this study there were no significant differences in the treatment effects of IFX, compared to the CP regarding VA, PU, AU and RV. RV became inactive after four months of treatment in all five patients in the IFX group versus four patients in the CP group.

Conclusion

IFX and CP have the same efficacy in the short-term treatment of uveitis in BD. In future investigations, multi-center studies with larger sample sizes are required to investigate the cost-effectiveness of IFX in the treatment of uveitis of BD.

Highlights

A new anti-TNF biologic agent, infliximab and cyclophosphamide have the same efficacy in the treatment of uveitis in BD, and since it has less toxicity, it is a superior treatment option.

In areas where access to the contemporary treatment is limited and expensive, the use of traditional medication results in an increased quality-adjusted life year (QALY).

Multi-center studies with larger sample sizes are required to investigate the cost-effectiveness of infliximab in the treatment of uveitis of BD.
The genetic landscape of kynurenine predicts neurovascular pathology and disrupted white matter integrity in patients with mood disorders
Authors: Bravi Beatrice , Fortanyer-Uyà Lidia , Paolini Marco , Comai Stefano , Poletti Sara , Cristina Lorenzi , Spadini Sara , Serretti Alessandro , Colombo Cristina , Zanardi Raffaella , Benedetti Francesco
Published: December 2024

Low-grade systemic inflammation is linked to cardiometabolic diseases and increased cardiovascular risk. Patients with mood disorders, such as Major Depressive Disorder (MDD) and Bipolar Disorder (BD), also show elevated cardiovascular risk and inflammatory markers, suggesting shared biological pathways between mood and cardiometabolic conditions. The kynurenine (KYN) pathway, activated by inflammatory cytokines and involved in neurotransmitter systems linked to mood, provides a promising area to explore inflammatory-related genetic overlaps in these disorders, with increasing interest in the SH2B3 rs3184504 SNP. Imaging markers like white matter hyperintensities (WMHs) and white matter (WM) microstructure alterations are associated with mood and cardiovascular disorders.

This study aimed to investigate the genetic load link to KYN levels, such as KYN polygenic risk score (PRS) and its effect on white matter hyperintensities (WMHs), outcomes of presumed vascular suffering, and WM microstructure in a sample of 95 MDD and 80 BD patients.

Higher PRS for KYN was associated with increased circulating KYN levels and KYN/TRP ratio. KYN PRS predicted the presence of WMHs. The SH2B3 rs3184504 T variant was associated with increased PRS for KYN and a higher number of WMHs. KYN levels and KYN/TRP ratio were not associated with WMHs, while KYN PRS positively correlated with higher axial (AD) and mean diffusivity (MD), with a nominal significance for radial diffusivity (RD).

The findings support a genetic contribution to elevated KYN and WM integrity alterations in mood disorders. PRS for KYN indicates a potential predisposition to inflammatory and vascular dysregulation, and SH2B3 rs3184504 may modulate this risk.
Perceived facilitators and barriers to physical activity, using the COM-B Model for Behavioural Change, in people with chronic pain: a qualitative evaluation of patient and stakeholder perspectives
Authors: Higgins C , Dall PM , Leese C , Adair P , Cameron PA , Inglis SK , Christogianni A , Smith BH , Colvin L
Published: December 2024

Background

Physical inactivity is a substantial public health concern and a particularly challenging problem to address. Physical activity is shown to be of therapeutic benefit to those living with chronic pain, but intervention studies have often been unsuccessful in promoting sustainable engagement with physical activity in this population.

Aim

To develop a more comprehensive understanding of the barriers to engaging with sustainable physical activity and the facilitators that promote this engagement in people with chronic pain.

Design & setting

The study design was qualitative, informed by behaviour change theory (the COM-B model), and participants were drawn from two settings. First, 36 adults living with moderate-severe chronic pain were recruited from a specialist NHS pain service. Secondly, 15 stakeholders were recruited to represent a range of healthcare sectors involved in supporting people with chronic pain.

Method

One-to-one semi-structured interviews, informed by the COM-B model, were conducted between May 2020 and October 2021. Interviews were conducted by telephone or using online conferencing software and were transcribedverbatimand anonymised. The Framework Method was used to interpret the data.

Results

The barriers to sustainable physical activity engagement in people with chronic pain are expansive, and the inclusion of stakeholder perspectives is valuable in developing a more comprehensive understanding of these barriers and also of the facilitators that are successful in promoting sustainable physical activity in this population.

Conclusions

The COM-B model provides a robust theoretical framework for organising and understanding these barriers and facilitators in a way could that inform effective intervention design.
COVID-19 vaccination and short-term mortality risk: a nationwide self-controlled case series study in The Netherlands
Authors: Slurink Isabel AL , de Boer Annemarijn R , Bonten Marc JM , Sturkenboom Miriam CJM , Bruijning-Verhagen PCJL
Published: December 2024

Excess mortality during the COVID-19 pandemic partly exceeded COVID-19-related deaths, indicating that other causes may have contributed. We conducted a retrospective data-linkage study including all Dutch inhabitants to investigate the impact of COVID-19 vaccination on excess mortality using a modified self-controlled case series method. We found a 44% lower relative incidence of all-cause deaths in the first three weeks after the primary vaccination compared to more than three weeks after vaccination (IRR 0.56, 95%CI 0.54-0.57). This lower incidence was consistent across vaccine types, doses, genders, age groups, and individuals with or without prior SARS-CoV-2 infection or comorbidities, and for non-COVID-19 related deaths. For booster vaccinations, the relative incidence was similar (IRR 0.49, 95%CI 0.49-0.50). In comparison, we observed a 16-fold higher incidence of all-cause deaths in the three weeks following a registered positive SARS-CoV-2 infection compared to more than three weeks after infection (IRR 16.19, 95%CI 15.78-16.60). A lower relative incidence of short-term deaths following COVID-19 vaccination support that COVID-19 vaccination is not associated with the observed excess mortality.
A pen mark is all you need - Incidental prompt injection attacks on Vision Language Models in real-life histopathology
Authors: Clusmann Jan , Schulz Stefan J. K. , Ferber Dyke , Wiest Isabella C. , Fernandez Aurélie , Eckstein Markus , Lange Fabienne , Reitsam Nic G. , Kellers Franziska , Schmitt Maxime , Neidlinger Peter , Koop Paul-Henry , Schneider Carolin V. , Truhn Daniel , Roth Wilfried , Jesinghaus Moritz , Kather Jakob N. , Foersch Sebastian
Published: December 2024

Vision-language models (VLMs) can analyze multimodal medical data. However, a significant weakness of VLMs, as we have recently described, is their susceptibility to prompt injection attacks. Here, the model receives conflicting instructions, leading to potentially harmful outputs. In this study, we hypothesized that handwritten labels and watermarks on pathological images could act as inadvertent prompt injections, influencing decision-making in histopathology. We conducted a quantitative study with a total of N = 3888 observations on the state-of-the-art VLMs Claude 3 Opus, Claude 3.5 Sonnet and GPT-4o. We designed various real-world inspired scenarios in which we show that VLMs rely entirely on (false) labels and watermarks if presented with those next to the tissue. All models reached almost perfect accuracies (90 - 100 %) for ground-truth leaking labels and abysmal accuracies (0 - 10 %) for misleading watermarks, despite baseline accuracies between 30-65 % for various multiclass problems. Overall, all VLMs accepted human-provided labels as infallible, even when those inputs contained obvious errors. Furthermore, these effects could not be mitigated by prompt engineering. It is therefore imperative to consider the presence of labels or other influencing features during future evaluation of VLMs in medicine and other fields.