bluuebunny/crossref_metadata_2025 huggingface dataset:
Label-free phenotyping of human microvessel networks
Authors: Rappez Luca , Akinbote Akinola , Cherubini Marta , Uhlmann Virginie , Haase Kristina
Published: February 2024

Understanding the spatial heterogeneity in blood vessel formation and development is crucial for various biomedical applications. Traditional methods forin-vitromicrovessel segmentation rely on fluorescent labeling, which either interferes with the sample homeostasis, limits the study to a restricted set of precursor cells, or requires sample fixation, thus preventing live measurements. Moreover, these methods often focus on small, cropped images, neglecting global spatial heterogeneity of microvasculature, leading to biased data interpretation. To overcome these limitations, we present VascuMap, a deep-learning-based tool for label-free vessel segmentation and spatial analysis. VascuMap enables a comprehensive examination of entire vessel networks, capturing both morphological and topological features across the full vascular bed. Our method achieves high segmentation accuracy, comparable to the state-of-the-art fluorescence-based models. VascuMap’s capabilities extend to characterizing vasculature generated from label-free patient-derived samples, a vital step towards personalized medicine. Its compatibility with widefield label-free microscopy also accelerates sample acquisition, making it ideal for high-throughput systems crucial for drug toxicity and safety screens.
A handle on mass coincidence errors inde novosequencing of antibodies by bottom-up proteomics
Authors: Schulte Douwe , Snijder Joost
Published: February 2024

Antibody sequences can be determined at 99% accuracy directly from the polypeptide product using bottom-up proteomics techniques. This circumvents the need to isolate the antibody-producing B-cell clone and enables reverse engineering of monoclonal antibodies from lost hybridoma cell lines, as well as the secreted protein in bodily fluid. Sequencing accuracy at the peptide level is limited by common mass coincidences of isobaric residues like leucine/isoleucine, but also by incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions. Likewise, different combinations of amino acids, of potentially different length, can also coincide to the same mass (e.g. GG=N, GA=Qetc.). Here we present several updates to Stitch (v1.5), which performs template-based assembly ofde novopeptide reads to reconstruct antibody sequences. This version introduces a mass-based alignment algorithm that explicitly accounts for mass coincidence errors. In addition, it incorporates a postprocessing procedure to assign I/L residues based on secondary fragments (satellite ions,i.e. w-ions). Moreover, evidence for sequence assignments can now be directly evaluated with the addition of an integrated spectrum viewer. This version of Stitch also allows input data from a wider selection ofde novopeptide sequencing algorithms, now including Casanovo, PEAKS, Novor.Cloud, pNovo, and MaxNovo, in addition to flat text and FASTA. Combined, these changes make Stitch compatible with a larger range of data processing pipelines and improve its tolerance to peptide-level sequencing errors.
Genomic 8-oxoguanine modulates gene transcription independent of its repair by DNA glycosylases OGG1 and MUTYH
Authors: Obermann Tobias , Sakshaug Teri , Kanagaraj Vishnu Vignesh , Abentung Andreas , Sarno Antonio , Bjørås Magnar , Scheffler Katja
Published: February 2024

8-oxo-7,8-dihydroguanine (OG) is one of the most abundant oxidative lesions in the genome and associated with genome instability. Its mutagenic potential is counteracted by a concerted action of 8-oxoguanine DNA glycosylase (OGG1) and mutY homolog DNA glycosylase (MUTYH). It has been suggested that OG and its repair has epigenetic-like properties and mediates transcription, but genome-wide evidence of this interdependence is lacking. Here, we applied an improved OG-sequencing approach reducing artificial background oxidation and RNA-sequencing to correlate genome-wide distribution of OG with gene transcription in OGG1 and/or MUTYH-deficient cells. Our data identified moderate enrichment of OG in the genome that is mainly dependent on the genomic context and not affected by DNA glycosylase-initiated repair. Interestingly, no association was found between genomic OG deposition and gene expression changes upon loss of OGG1 and MUTYH. Regardless of DNA glycosylase activity, OG in promoter regions correlated with expression of genes related to metabolic processes and damage response pathways indicating that OG functions as a cellular stress sensor to regulate transcription. Our work provides novel insights into the mechanism underlying transcriptional regulation by OG and DNA glycosylases OGG1 and MUTYH and suggests that oxidative DNA damage accumulation and its repair utilize different pathways.
Communication with Surprise – Computational Principles of Goal Signaling in Novel Human Interactions
Authors: Buidze Tatia , Sommer Tobias , Zhao Ke , Fu Xiaolan , Gläscher Jan
Published: February 2024

Communication, often grounded in shared expectations, faces challenges without common linguistic backgrounds. Our study explores how people instinctively turn to the fundamental principles of the physical world to overcome communication barriers. Specifically, through the Tacit Communication Game, we investigate how participants develop novel strategies for conveying messages without relying on common linguistic signals. We developed a new computational model built from the principle of expectancy violations of a set of common universal priors derived from movement kinetics. The model closely resembles the Sender’s messages, with its core variable – the information-theoretic surprise – explaining the Receiver’s physiological and neural responses. This is evidenced by a significant correlation with the pupil diameter, indicating cognitive effort, and neural activity in brain areas related to expectancy violations. This work highlights the adaptability of human communication, showing how surprise can be a powerful tool in forming new communicative strategies without relying on common language.
Optic nerve injury impairs intrinsic mechanisms underlying electrical activity in a resilient retinal ganglion cell
Authors: Zapadka Thomas E. , Tran Nicholas M. , Demb Jonathan B.
Published: February 2024

Retinal ganglion cells (RGCs) are the sole output neurons of the retina and convey visual information to the brain via their axons in the optic nerve. Following an injury to the optic nerve, RGCs axons degenerate and many cells die. For example, a surgical model of compressive axon injury, the optic nerve crush (ONC), kills ∼80% of RGCs after two weeks. Surviving cells are biased towards certain ‘resilient’ types, including several types that originally produced sustained firing to light stimulation. RGC survival may depend on activity level, and there is a limited understanding of how or why activity changes following optic nerve injury. Here we quantified the electrophysiological properties of a highly resilient RGC type, the sustained ON-Alpha RGC, seven days post-ONC with extracellular and whole-cell patch clamp recording. Both light- and current-driven firing were reduced after ONC, but synaptic inputs were largely intact. Resting membrane potential and input resistance were relatively unchanged, while voltage-gated currents were impaired, including a reduction in voltage-gated sodium channel density in the axon initial segment and function. Hyperpolarization or chelation of intracellular calcium partially rescued firing rates. These data suggest that an injured resilient RGC reduces its activity by a combination of reduced voltage-gated channel expression and function and downregulation of intrinsic excitability via a Ca2+-dependent mechanism without substantial changes in synaptic input. Reduced excitability may be due to degradation of the axon but could also be energetically beneficial for injured RGCs, preserving cellular energy for survival and regeneration.

Graphical Abstract

Schematic view of the effects of axon injury (optic nerve crush) on the physiology of an sustained ON-Alpha (AlphaONS) retinal ganglion cell. These cells are highly resilient to axon injury and survive for several weeks while other retinal ganglion cell types perish. At one week after injury, the AlphaONS RGC has diminished spontaneous and light-evoked action potential firing. Reduced firing depends not on changes in synaptic inputs but rather on reductions in intrinsic excitability. Reduced excitability is explained by a Ca2+-dependent mechanism and by a reduction in sodium channel density and function.

Key Points Summary

1)

Retinal ganglion cell (RGC) types show diverse rates of survival after axon injury.

2)

A resilient RGC type maintains its synaptic inputs one week post-injury.

3)

The resilient RGC type shows diminished firing and reduced expression of axon initial segment (AIS) genes following injury

4)

Activity deficits arise from intrinsic dysfunction (Na+channels, intracellular Ca2+), not from loss of excitation or enhanced inhibition.
Rejuvenating silicon probes for acute electrophysiology
Authors: Shoup Alden M. , Porwal Natasha , Fakharian Mohammad Amin , Hage Paul , Orozco Simon P. , Shadmehr Reza
Published: February 2024

Electrophysiological recording with a new probe often yields better signal quality than with a used probe. Why does the signal quality degrade after only a few experiments? Here, we considered silicon probes in which the contacts are densely packed, and each is coated with a conductive polymer that increases its surface area. We tested 12 Cambridge Neurotech silicon probes during 61 recording sessions from the brain of 3 marmosets. Out of the box, each probe arrived with an electrodeposited polymer coating on 64 gold contacts, and an impedance of around 50k Ohms. With repeated use, the impedance increased and there was a corresponding decrease in the number of well-isolated neurons. Imaging of the probes suggested that the reduction in signal quality was due to a gradual loss of the polymer coating. To rejuvenate the probes, we first stripped the contacts, completely removing their polymer coating, and then recoated them in a solution of 10 mM EDOT monomer with 32 uM PSS using a current density of about 3mA/cm2for 30 seconds. This recoating process not only returned probe impedance to around 50k Ohms, it also yielded significantly improved signal quality during neurophysiological recordings. Thus, insertion into the brain promoted loss of the polymer that coated the contacts of the silicon probes. This led to degradation of signal quality, but recoating rejuvenated the probes.
Biomaterial Degradation Products affect Regenerating Glia Independently of Surface Properties
Authors: Pawelec Kendell M , Hix Jeremy ML , Shapiro Erik M
Published: February 2024

Devices to treat peripheral nerve injury (PNI) must balance many considerations to effectively guide regenerating nerves across a gap and achieve functional recovery. To enhance efficacy, design features like luminal fillers have been explored extensively. Material choice for PNI devices is also critical, as the determining factor of device mechanics, and degradation rate and has increasingly been found to directly impact biological response. This study investigated the ways in which synthetic polymer materials impact the differentiation state and myelination potential of Schwann cells, peripheral nerve glia. Microporous substrates of polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15, or PLGA 50:50 were chosen, as materials already used in nerve repair devices, representing a wide range of mechanics and degradation profiles. Schwann cells co-cultured with dorsal root ganglion (DRG) neurons on the substrates expressed more mature myelination proteins (MPZ) on PLGA substrates compared to PCL. Changes to myelination and differentiation state of glia were reflected in adhesion proteins expressed by glia, including β-dystroglycan and integrin α6, both laminin binding proteins. Importantly, degradation products of the polymers affected glial expression independently of direct attachment. Fast degrading PLGA 50:50 substrates released measurable amounts of degradation products (lactic acid) within the culture period, which may push Schwann cells towards glycolytic metabolism, decreasing expression of early transcription factors like sox10. This study shows the importance of understanding not only material effects on attachment, but also on cellular metabolism which drives myelination responses.
Empowering high-throughput high-content analysis of 3D tumor models: Open-source software for automated non-confocal image analysis
Authors: Wiggin Noah , Cook Carson , Black Mitchell , Cadena Ines , Rahal-Arabi Salam , Fogg Kaitlin
Published: February 2024

Purpose

The primary aim of this study was to develop an open-source Python-based software for the automated analysis of dynamic cell behaviors in three-dimensional tumor models using non-confocal microscopy. This research seeks to address the existing gap in accessible tools for high-throughput analysis of cancer and endothelial cell dynamicsin vitro, facilitating the rapid assessment of drug sensitivity.

Methods

Our approach involved annotating over 1000 2 mm Z-stacks of cancer and endothelial cell co-culture model and training machine learning models to automatically calculate cell coverage, cancer invasion depth, and microvessel dynamics. Specifically, cell coverage area was computed using focus stacking and Gaussian mixture models to generate thresholded Z-projections. Cancer invasion depth was determined using a deep neural network binary classification model, measuring the distance between Z-planes with invaded cells. Lastly, microvessel dynamics were assessed through a U-Net Xception-style deep learning model for segmentation, a disperse algorithm for network graph representation, then persistent homology to quantify microvessel length and connectivity. Finally, we reanalyzed an image set from a high-throughput drug screen involving a chemotherapy agent on a 3D cervical and endothelial co-culture model.

Results

The software accurately measured cell coverage, cancer invasion, and microvessel length, yielding drug sensitivity IC50values with a 95% confidence level compared to manual calculations. Additionally, it significantly reduced the image processing time from weeks down to hours.

Conclusions

Our free and open source software offers an automated solution for quantifying 3D cell behavior in tumor models using non-confocal microscopy, providing the broader Cellular and Molecular Bioengineering community with an alternative to standard confocal microscopy paired with proprietary software.
Normative and individual, non-normative intrinsic networks and the transition to impaired cognition
Authors: Zhang Qirui , Hudgins Stacy , Struck Aaron F , Ankeeta Ankeeta , Javidi Sam S. , Sperling Michael R , Hermann Bruce P , Tracy Joseph I.
Published: February 2024

Despite their temporal lobe pathology, a significant subgroup of temporal lobe epilepsy (TLE) patients are able to maintain normative cognitive functioning. Here, we identify TLE patients with intact versus impaired cognitive profiles, and interrogate for the presence of both normative and highly individual intrinsic connectivity networks(ICN) – all towards understanding the transition from impaired to intact neurocognitive status. We retrospectively investigated data from 88 TLE patients and matched 91 healthy controls with resting-state functional MRI. Functional MRI data were decomposed using independent component analysis to obtain individualized ICNs. Here, we calculated the degree of match between individualized ICNs and canonical ICNs (e.g., Yeo et.al 17 resting-state network) and divided each participant’s ICNs into normative or non-normative status based on the degree of match. We found that the individualized networks matched the canonical networks less well in the cognitively impaired compared to the cognitively intact TLE patients. The cognitively impaired patients showed significant abnormalities in the profiles of both normative and non-normative networks, whereas the intact patients showed abnormalities only in non-normative networks. At the same time, we found normative networks held a strong, positive association with the neuropsychological measures, with this association negative in non-normative networks. We were able to provide the initial data demonstrating that significant cognitive deficits are associated with the status of highly-individual ICNs, making clear that the transition from intact to impaired cognitive status is not simply the result of disruption to normative brain networks.
Integrating short-read and long-read single-cell RNA sequencing for comprehensive transcriptome profiling in mouse retina
Authors: Wang Meng , Li Yumei , Wang Jun , Oh Soo Hwan , Chen Rui
Published: February 2024

The vast majority of protein-coding genes in the human genome produce multiple mRNA isoforms through alternative splicing, significantly enhancing the complexity of the transcriptome and proteome. To establish an efficient method for characterizing transcript isoforms within tissue samples, we conducted a systematic comparison between single-cell long-read and conventional short-read RNA sequencing techniques. The transcriptome of approximately 30,000 mouse retina cells was profiled using 1.54 billion Illumina short reads and 1.40 billion Oxford Nanopore long reads. Consequently, we identified 44,325 transcript isoforms, with a notable 38% previously uncharacterized and 17% expressed exclusively in distinct cellular subclasses. We observed that long-read sequencing not only matched the gene expression and cell-type annotation performance of short-read sequencing but also excelled in the precise identification of transcript isoforms. While transcript isoforms are often shared across various cell types, their relative abundance shows considerable cell-type-specific variation. The data generated from our study significantly enhance the existing repertoire of transcript isoforms, thereby establishing a foundational resource for future research into the mechanisms and implications of alternative splicing within retinal biology and its links to related diseases.
Antimicrobial resistance, virulence genes profiles and molecular epidemiology of carbapenem-resistantKlebsiella pneumoniaestrains from captive giant pandas (Ailuropoda melanoleuca)
Authors: Yan Xia , Yang Mei , Ayala James Edward , Li Lin , Zhou Yang , Hou Rong , Liu Songrui , Li Yunli , Yue Chanjuan , Zhang Dongsheng , Su Xiaoyan
Published: February 2024

This study aimed to investigate the antibiotic susceptibility, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), virulence genes, and molecular epidemiology of carbapenem-resistantKlebsiella pneumoniae(CRKP) strains isolated from giant pandas. The screening of 178 nonduplicatedKlebsiella pneumoniaestrains identified eight CRKP strains, with the most abundant ARGs observed in ampC/blaDHA, blaSHV-01, blaSHV-02, tetB-01, tetB-02, tetC-01, and tetC-02. MGE analysis revealed the presence of intI1 in all strains, while other MGEs exhibited varying detection rates. Strain 24 exhibited the highest diversity in terms of MGE species. Seven virulence genes including wabG, uge, ycf, entB, kpn, alls, and wcaG, showed positive results with different proportions across the strains. Molecular epidemiology analysis using pulsed-field gel electrophoresis (PFGE) patterns indicated a high level of genetic diversity among the CRKP strains. Multi-locus sequence typing (MLST) analysis classified the strains into different sequence types (STs). In conclusion, this study highlighted the diverse nature of CRKP strains found in giant pandas, which exhibited varying levels of antibiotic resistance along with multiple ARGs and virulence genes present. These findings emphasized the importance of monitoring and researching antibiotic resistance within wildlife populations to safeguard the health status of these endangered animals.
The 2023 wildfire season in Québec: an overview of extreme conditions, impacts, lessons learned and considerations for the future
Authors: Boulanger Yan , Arseneault Dominique , Bélisle Annie Claude , Bergeron Yves , Boucher Jonathan , Boucher Yan , Danneyrolles Victor , Erni Sandy , Gachon Philippe , Girardin Martin P. , Grant Eliane , Grondin Pierre , Jetté Jean-Pierre , Labadie Guillemette , Leblond Mathieu , Leduc Alain , Puigdevall Jesus Pascual , St-Laurent Martin-Hugues , Tremblay Junior A. , Waldron Kaysandra
Published: February 2024

The 2023 wildfire season in Québec set records due to extreme warm and dry conditions, burning 4.5 million hectares and indicating persistent and escalating impacts associated with climate change. The study reviews the unusual weather conditions that led to the fires, discussing their extensive impacts on the forest sector, fire management, boreal caribou habitats, and particularly the profound effects on First Nation communities. The wildfires led to significant declines in forest productivity and timber supply, overwhelming fire management resources, and necessitating widespread evacuations. First Nation territories were dramatically altered, facing severe air quality issues and disruptions. While caribou impacts were modest across the province, the broader ecological, economical, and social repercussions were considerable. To mitigate future extreme wildfire seasons, the study suggests changes in forest management practices to increase forest resilience and resistance, adapting industrial structures to new timber supplies, and enhancing fire suppression and risk management strategies. It calls for a comprehensive, unified approach to risk management that incorporates the lessons from the 2023 fire season and accounts for ongoing climate change. The study underscores the urgent need for detailed planning and proactive measures to reduce the growing risks and impacts of wildfires in a changing climate.
Antidiabetic effect of 1-deazapurines derivatives with alpha-glucosidase Enzyme: A molecular tools approach
Authors: Hacene Faiza Boukli , Cherrak Sabri Ahmed , Soufi Wassila , Ghalem Said
Published: February 2024

Alpha-glucosidase inhibition has been shown by several 1-deazapurine derivatives that have already been synthesized and evaluated in vitro, providing a potential treatment target for type 2 diabetes. Six of them were shown to have higher percentages of inhibition against the alpha-glucosidase enzyme and lower IC50 values. The binding mechanism and stability of the generated complexes are investigated in the current work using various molecular modeling methodologies. The ligands L17, L11, and L4 present the best binding energy with the establishment of interaction toward the site active, based on the results of the molecular docking simulation. The stability of the chosen complexes was then validated using molecular dynamics simulation. However, ADME-T prediction and Drug-likeness results show that these compounds have promising pharmacokinetic properties and oral bioavailability. Finally, these results imply that compounds L4 and L11 are very promising as a target for creating a lead molecule for type 2 diabetes.
Genomic localization bias of secondary metabolite gene clusters and association with histone modifications inAspergillus
Authors: Zhang Xin , Leahy Iseult , Collemare Jérȏme , Seidl Michael F.
Published: February 2024

Fungi are well-known producers of bioactive secondary metabolites (SMs), which have been exploited for decades by humankind for various medical applications like therapeutics and antibiotics. SMs are synthesized by biosynthetic gene clusters (BGCs) – physically co-localized and co-regulated genes. Because BGCs are often regulated by histone post-translational modifications (PTMs), it was suggested that their chromosomal location is important for their expression. Studies in a few fungal species indicated an enrichment of BGCs in sub-telomeric regions; however, there is no evidence that BGCs with distinct genomic localization are regulated by different histone PTMs. Here, we used 174Aspergillusspecies covering 22 sections to determine the correlation between BGC genomic localization, gene expression and histone PTMs. We found a high abundance and diversity of SM backbone genes across theAspergillusgenus, with notable unique genes within sections. Being unique or conserved in many species, BGCs showed a strong bias for being localized in low-synteny regions, regardless of their position in chromosomes. Using chromosome-level assemblies, we also confirmed a significantly biased localization in sub-telomeric regions. Notably, SM backbone genes in sub-telomeric regions and about half of those in low-synteny regions exhibit higher gene expression variability, likely due to the similar higher variability in H3K4me3 and H3K36me3 histone PTMs; while variations in histone H3 acetylation and H3K9me3 are not correlated to genomic localization and expression variation, as analyzed in twoAspergillusspecies. Expression variability across fourAspergillusspecies further supports that BGCs tend to be located in low-synteny regions and that regulation of expression in those regions likely involves different histone PTMs than the most commonly studied modifications.

Significance

Fungi are known for producing an array of bioactive compounds with medical benefits, yet our understanding of how the production of these compounds is regulated remains limited. Here, we focused on the fungal genusAspergillus, containing many species known to be prolific producers of bioactive compounds, to systematically uncover the diversity and genomic localization of biosynthetic pathways. By expanding our knowledge beyond the few commonly studied fungal species, this research offers novel insights into how the genomic localization of biosynthetic pathways matters for the regulation of their expression. Thanks to a new view on BGC localization and expression in relation to histone modifications, our results are expected to stimulate functional research on neglected histone modifications that will support the discovery and harnessing of new fungal metabolites for medical and industrial applications.
Infant neural sensitivity to affective touch is associated with maternal postpartum depression
Authors: Nance Madelyn G. , Landsman Zackary T. , Gerling Gregory J , Puglia Meghan H.
Published: February 2024

Classic attachment theory emphasizes the sensitivity of the parent to perceive and appropriately respond to the infant's cues. However, parent-child attachment is a dyadic interaction that is also dependent upon the sensitivity of the child to the early caregiving environment. Individual differences in infant sensitivity to parental cues is likely shaped by both the early caregiving environment as well as the infant's neurobiology, such as perceptual sensitivity to social stimuli. Here, we investigated associations between maternal postpartum depression and infant neurological sensitivity to affective touch using brain signal entropy - a metric of the brain's moment-to-moment variability related to signal processing. We recruited two independent samples of infants aged 0-5 months. In Sample 1 (n=79), we found increased levels of maternal postpartum depression were associated with diminished perceptual sensitivity - i.e. lower entropy - to affective tactile stimulation specifically within the primary somatosensory cortex. In Sample 2 (n=36), we replicated this finding and showed that this effect was not related to characteristics of the touch administered during the experiment. These results suggest that decreased affective touch early in life - a common consequence of postpartum depression - likely impacts the infant's perceptual sensitivity to affective touch and ultimately the formation of experience-dependent neural networks that support the successful formation of attachment relationships.
Imaging and quantification of prostate cancer-associated bone by polarization-sensitive optical coherence tomography
Authors: Zhou Chris , Jung Naomi , Xu Samuel , Eltit-Guersetti Felipe , Lu Xin , Wang Qiong , Liang Doris , Morrissey Colm , Corey Eva , True Lawrence D. , Wang Rizhi , Tang Shuo , Cox Michael E.
Published: February 2024

Prostate cancer frequently metastasizes to bone, leading to a spectrum of osteosclerotic and osteolytic lesions that cause debilitating symptoms. Accurate differentiation of bone tissue or lesion types can provide opportunity for local pathologic investigation, which is critical for understanding the bone metastasis and remains challenging. Current imaging methods lack the ability to directly differentiate tissues based on collagen organization and may induce invasive effect on bone tissues. We introduce Polarization-Sensitive Optical Coherence Tomography (PS-OCT) to investigate normal, osteosclerotic, and osteolytic bone tissues. High-resolution PS-OCT imaging reveals collagen fiber arrangement, enabling nuanced distinction of degree of collagen alignment among different bone tissue types or regions. We present a novel feature named degree of ordered organization (DOO), derived from the multiple contrasts of PS-OCT that can quantitatively evaluate bone samples from different pathologic groups, including control,osteoblastic and osteolytic tissues. The capacity of PS-OCT to differentiate trabecular/lamellar and irregular (woven bone) regions within the same specimen is tested and validated on ex-vivo samples extracted from 13 subjects. Our study is the first time that PS-OCT is applied to metastatic bone disease with the aim of enhancing the understanding of bone-related pathologies, and potentially impacting clinical practice. This work demonstrates that PS-OCT can provide useful insight into bone microstructures, and thus it has potential applications across diverse bone disorders.
Arginine Di-methylation of RIPK3 Safeguards Necroptosis for Intestinal Homeostasis
Authors: Zhao Pan , Dan Hanjun , Wang Yazhou , Chen Xin , Jiang Xiangling , Shen Yao , Wang Jiajia , Yang Zhiwei , Zhao Jiasheng , Zhang Yingying , Zheng Jianyong , Liu Wen , Zhang Jian
Published: February 2024

The necroptosis mediated by RIPK3 is stringently regulated for intestinal homeostasis. Here we found that mice lackingPrmt5(Protein arginase methyltransferase 5) in intestinal epithelial cells (IECs) caused premature death with IECs necroptosis, villus atrophy and loss of Paneth cells. This pathology can be partially rescued by antibiotic treatment, germ-free breeding condition and pharmaceutical inhibition of RIPK1 and RIPK3, but aggravated for embryonic lethality byCaspase-8 deficiency, which demonstrating the importance of commensal bacteria and necroptosis for thePrmt5-IEC deficiency. Intriguingly, tumor-necrosis factor (TNF) receptor 1(Tnfr1) deficiency could not completely rescue the pathology, and mice deficit in Z- DNA binding protein 1(ZBP1) exhibited shorter lifespan compared withPrmt5null mice, suggestingPrmt5loss might trigger TNFR-RIPK1-depenfent and ZBP1- dependent necroptosis. Mechanically, we identified the 479-arginine residue of RIPK3 di-methylated by PRMT5 was an endogenous checkpoint for necroptosis. Furthermore, RIPK3-R479K mutation had higher affinity with both RIPK1 and ZBP1 by immunoprecipitation and STORM (Stochastic Optical Reconstruction Microscopy) analysis, which might explain the endogenous necroptosis triggered by mutated RIPK3 even without upstream stimuli. Moreover, the peptide of RIPK3-SDMA (Symmetric dimethylarginine of 479) could rescue lethality ofPrmt5 lacking mice through necrosome formation inhibition, which demonstrating the great potential for necroptosis-related disease treatment through RIPK3 dimethylation targeting.
CanLncG4: A database curated for the assessment of G4s in the lncRNAs dysregulated in various human cancers
Authors: Sharma Shubham , Yusuf Muhammad , Barbhuiya Noman Hasif , Ramolia Harshit , Shukla Chinmayee , Singh Deepshikha , Datta Bhaskar
Published: February 2024

Long non-coding RNAs (lncRNAs) comprise a substantive part of the human genome and have emerged as crucial participants of cellular processes and disease pathogenesis. Dysregulated expression of lncRNAs in cancer contributes to various hallmarks of the disease, presenting novel opportunities for diagnosis and therapy. G-quadruplexes (G4s) within lncRNAs have gained attention, though their systematic evaluation in cancer biology is yet to be performed. In this work, we have formulated CanLncG4, a comprehensive database integrating experimentally validated associations between lncRNAs and cancer, and detailed predictions of their G4-forming potential. CanLncG4 categorizes predicted G4 motifs into anticipated G4 types and offers insights into the subcellular localization of the corresponding lncRNAs. It provides information on lncRNA-RNA and lncRNA-protein interactions, together with the RNA G4-binding capabilities of these proteins. To ensure the accuracy and validity of the data sourced from various databases, a meticulous examination of the output data was conducted to identify any discrepancies, including incorrect, missing, or duplicate entries. Additionally, scientific literature mining was performed to cross-validate the gathered information. Data from G4-prediction tools was generated using multiple parameter combinations to determine the parameters that yield more relevant and accurate predictions of the G4-forming potential. We validate ourin silicoG4-prediction pipeline throughin vitroexperiments, affirming the presence of G4s within specific cancer-dysregulated lncRNAs, thereby illustrating the predictive capability of CanLncG4. CanLncG4 represents a valuable resource for investigating G4-mediated lncRNA functions in diverse human cancers. It is expected to provide distinctive leads about G4-mediated lncRNA-protein interactions. CanLncG4 comprehensively documents 17,666 entries, establishing correlations between 6,408 human lncRNAs encompassing their transcript variants, and 15 distinct types of human cancers. The database is freely available athttps://canlncg4.com/, offering researchers a valuable tool for exploring lncRNA and G4 biology towards cancer diagnosis and therapeutics.
Regionalized cell and gene signatures govern oesophageal epithelial homeostasis
Authors: Grommisch David , Lund Harald , Eenjes Evelien , Julien Anais , Göritz Christian , Harris Robert A. , Sandberg Rickard , Hagemann-Jensen Michael , Genander Maria
Published: February 2024

Regionalized disease prevalence is a common feature of the gastrointestinal tract. Herein, we employed regionally resolved Smart-seq3 single-cell sequencing, generating a comprehensive cell atlas of the adult mouse oesophagus. Characterizing the oesophageal axis, we unveil non-uniform distribution of epithelial basal cells, fibroblasts and immune cells. In addition, we reveal a position-dependent, but cell subpopulation-independent, transcriptional signature, collectively generating a regionalized oesophageal landscape.

Combiningin vivomodels with organoid co-cultures, we demonstrate that proximal and distal basal progenitor cell states are functionally distinct. We find that proximal fibroblasts are more permissive for organoid growth compared to distal fibroblasts and that the immune cell profile is regionalized in two dimensions, where proximal-distal and epithelial-stromal gradients impact epithelial maintenance. Finally, we predict and verify how WNT-, BMP-, IGF-and NRG-signalling are differentially engaged along the oesophageal axis.

We establish a cellular and transcriptional framework for understanding oesophageal regionalization, providing a functional basis for epithelial disease susceptibility.
Mucin MUC5B levels are reduced in the colonic epithelia of ulcerative colitis patients
Authors: Ding Wei , Koltun Walter A. , Yochum Gregory S.
Published: February 2024

Background & Aims

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) where a defect in the colonic epithelial barrier elicits a chronic and unresolved inflammatory response against luminal microbiota. Mucins are heavily glycosylated proteins secreted by goblet cells that form gelatinous layers to protect the colonic epithelium. Whereas the role of the major colonic mucin, MUC2, is established in UC, few reports have analyzed MUC5B in this disease. In this study, we investigated MUC5B expression in colonic tissues and organoid cultures from UC patients and non-IBD controls.

Methods

MUC5Btranscripts were analyzed in colonic regions from UC patients and controls using RT-qPCR. MUC5B protein levels in colonic tissues were analyzed and quantified by immunohistochemistry. Patient-derived epithelial organoids were stained for MUC5B, E-cadherin and F-actin. Organoids were treated with interleukin 1 beta (IL-1β) andMUC5Btranscripts were analyzed by RT-qPCR. MUC5B proteins were assessed in IL-1β-treated organoids by immunofluorescent microscopy.

Results

MUC5Btranscripts and proteins were expressed in the healthy colon and their levels were decreased in UC tissues. MUC5B proteins were reduced in organoids derived from UC tissues. IL-1β treatment of control organoids stimulated expression of acidic mucins,MUC5Btranscripts and MUC5B proteins.MUC5Btranscripts were not induced by IL-1β in UC-derived organoids.

Conclusions

MUC5B levels are reduced in UC colons and its expression is induced by the IL-1β pro-inflammatory cytokine. These results suggest that MUC5B contributes to the protective colonic mucin layer and that this function is likely compromised in UC.
diaPASEF Enables High-throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development
Authors: Tsukidate Taku , Stiving Alyssa Q. , Rivera Shannon , Sahoo Ansuman , Madabhushi Sri , Li Xuanwen
Published: February 2024

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutics production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography–mass spectrometry (LC–MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS–based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein, as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation–serial fragmentation combined with data-independent acquisition (diaPASEF), to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.
The wide spectrum anti-inflammatory activity of andrographolide in comparison to NSAIDs: a promising therapeutic compound against the cytokine storm
Authors: Low Mitchell , Suresh Harsha , Zhou Xian , Bhuyan Deep Jyoti , Alsherbiny Muhammad A. , Khoo Cheang , Münch Gerald , Li Chun Guang
Published: February 2024

The challenges of the COVID-19 pandemic have highlighted an increasing clinical demand for safe and effective treatment options against an overzealous immune defence response, also known as the “cytokine storm”. Andrographolide is a naturally derived bioactive compound with promising anti-inflammatory activity in many clinical studies. However, its cytokine-inhibiting activity, in direct comparison to commonly used nonsteroidal anti-inflammatory drugs (NSAIDs), has not been extensively investigated in existing literature. The anti-inflammatory activities of andrographolide and common NSAIDs, such as diclofenac, aspirin, paracetamol and ibuprofen were measured on lipopolysaccharide (LPS) and interferon-γ induced RAW264.7 cells. The levels of PGE2, nitric oxide (NO), TNF-α & LPS-induced release of pro-inflammatory cytokines on differentiated human macrophage THP-1 cells were measured against increasing concentrations of andrographolide and aforementioned NSAIDs. The associated mechanistic pathway was examined on NFκB using flow cytometry on the human endothelial-leukocyte adhesion molecule (ELAM9) (E-selectin) transfected RAW264.7 cells with green fluorescent protein (GFP). Andrographolide exhibited broad and potent anti-inflammatory and cytokine-inhibiting activity in both cell lines by inhibiting the release of IL-6, TNF-α and IFN-γ, which are known to play a key role in the etiology of cytokine storm and the pathogenesis of inflammation. In comparison, the tested NSAIDs demonstrated weak or no activity against proinflammatory mediators except for PGE2, where the activity of andrographolide (IC50= 8.8 µM, 95% CI= 7.4 to 10.4 µM) was comparable to that of paracetamol (IC50= 7.73 µM, 95% CI = 6.14 to 9.73 µM). The anti-inflammatory action of andrographolide was associated with its potent downregulation of NFκB. The wide-spectrum anti-inflammatory activity of andrographolide demonstrates its therapeutic potential against cytokine storms as an alternative to NSAIDs.
Metabolicshift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells
Authors: Eduardo Mariana Bustamante , Cottone Gannon , McCloskey Curtis W. , Liu Shiyu , Zappia Maria Paula , Benevolenskaya Elizaveta V. , Islam A.B.M.M.K. , Frolov Maxim V. , Palma Flavio R. , Gao Peng , Setya Joel , Gao Hongyu , Xuei Xiaoling , Khokha Rama , Locasale Jason , Bonini Marcelo G. , Chandel Navdeep S. , Khan Seema A. , Clare Susan E.
Published: February 2024

A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increasing flux through various metabolic reactions; but little is known about the mechanism(s) linking lipids and epigenetic reprogramming with the genesis of ER-BC. Here we show that the metabolism of the medium-chain fatty acid Octanoic Acid (OA) in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM, glutathione, and 2-HG, with implications for oncogenesis: SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express Neural, EMT and BC related genes. 2-HG exposure results in appearance of DNA breaks, potentially consequent to the inhibition of essential demethylases for HR repair. ROS increases shortly after OA exposure and is mitigated by antioxidant defenses, which favors/enables the survival of specific cell subtypes.
Identification and characterization of a skin microbiome onCaenorhabditis eleganssuggests environmental microbes confer cuticle protection
Authors: Haghani Nadia , Lampe Robert H. , Samuel Buck , Chalasani Sreekanth H. , Matty Molly A.
Published: February 2024

In the wild,C. elegansare emersed in environments teeming with a veritable menagerie of microorganisms. TheC. eleganscuticular surface serves as a barrier and first point of contact with this microbial milieux. Here, we identify microbes fromC. elegansnatural habitats that associate with its cuticle, constituting a simple ‘skin microbiome’. We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on theC. eleganssurface. Further, we conduct 16S rRNA gene sequencing studies to identify relative shifts in the proportion of mCeMbio bacteria upon surface-sterilization, implying distinct skin-and gut-microbiomes. We find that some strains of bacteria, includingEnterobacter sp.JUb101, are primarily found on the nematode skin, while others likeStenotrophomonas indicatrixJUb19 andOchrobactrum vermisMYb71 are predominantly in the animal’s gut. Finally, we show that this skin microbiome promotes host cuticle integrity in harsh environments. Together, we identify a skin microbiome for the well-studied nematode model and propose its value in conferring host fitness advantages in naturalized contexts.

Importance

The genetic model organismC. eleganshas recently emerged as a tool for understanding host-microbiome interactions. Nearly all of these studies either focus on pathogenic or gut-resident microbes. Little is known about the existence of native, non-pathogenic skin microbes or their function. We show that members of a modifiedC. elegansmodel microbiome, mCeMbio, can adhere to the animal’s cuticle and confer protection from noxious environments. We combine a novel micro-swab tool, the first 16S microbial sequencing data from relatively unperturbedC. elegans, and physiological assays to demonstrate microbially mediated protection of the skin. This work serves as a foundation to explore wildC. elegansskin microbiomes and useC. elegansas a model for skin research.
Antagonistic Roles of Tau and MAP6 in Regulating Neuronal Development
Authors: Sun Xiaohuan , Yu Wenqian , Baas Peter W. , Toyooka Kazuhito , Qiang Liang
Published: February 2024

Association of tau with microtubules causes them to be labile while association of MAP6 with microtubules causes them to be stable. As axons differentiate and grow long, tau and MAP6 segregate from one another on individual microtubules, resulting in the formation of stable and labile domains. The functional significance of the yin/yang relationship between tau and MAP6 remained speculative in those studies, with one idea being that such a relationship assists in balancing morphological stability with plasticity. Here, using primary rodent neuronal cultures, we show that depletion of tau has opposite effects compared to depletion of MAP6 on the rate of neuronal development, the efficiency of growth cone turning, and the number of processes and axonal branches. Opposite effects to those of tau depletion were also observed on the rate of neuronal migration, in anin vivoassay, when we depleted MAP6. When tau and MAP6 were depleted together in the cell culture assays, the morphological phenotypes negated one another. Tau and MAP6 are multifunctional proteins, but the present results suggest that the observed effects of their depletion on neuronal development are likely due to their opposite roles in regulating microtubule dynamics.

Tau and MAP6 play antagonistic roles in regulating multiple aspects of neuronal development, presumably via their antagonistic effects on microtubule dynamics.
CYpHER: Catalytic extracellular targeted protein degradation with high potency and durable effect
Authors: Crook Zachary R. , Sevilla Gregory P. , Young Pamela , Girard Emily J. , Phi Tinh-Doan , Howard Monique , Price Jason , Olson James M. , Nairn Natalie W.
Published: February 2024

Many disease-causing proteins have multiple pathogenic mechanisms, and conventional inhibitors struggle to reliably disrupt more than one. Targeted protein degradation (TPD) can eliminate the protein, and thus all its functions, by directing a cell’s protein turnover machinery towards it. Two established strategies either engage catalytic E3 ligases or drive uptake towards the endolysosomal pathway. Here we describe CYpHER (CatalYticpH-dependentEndolysosomal delivery withRecycling) technology with potency and durability from a novel catalytic mechanism that shares the specificity and straightforward modular design of endolysosomal uptake. By bestowing pH-dependent release on the target engager and using the rapid-cycling transferrin receptor as the uptake receptor, CYpHER induces endolysosomal target delivery while re-using drug, potentially yielding increased potency and reduced off-target tissue exposure risks. The TfR-based approach allows targeting to tumors that overexpress this receptor and offers the potential for transport to the CNS. CYpHER function was demonstratedin vitrowith EGFR and PD-L1, andin vivowith EGFR in a model of EGFR-driven non-small cell lung cancer.