bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational changes in the AdeB transmembrane efflux pump by amphiphilic peptide Mastoparan-B, down-regulates expression of theadeB Gene and restores antibiotics Susceptibility
Authors: Shakibaie Mohammad Reza , Modaresi Farzan , Azizi Omid , Tadjrobehkar Omid , Ghaemi Mohammad Mehdi
Published: January 2023

No report exists on the role of Mastoparan B (MP-B) as an RND efflux pump inhibitor in multi-drug resistant (MDR)Acinetobacter baumannii. Here, we performed a series of in-silico experiments to predict the inhibition of the AdeB efflux pump by MP-B as a drug target agent. For this reason, an MDR strain ofA. baumanniiwas subjected to MICs against 12 antibiotics as well as MP-B. Expression of the adeB gene in the presence and absence of sub-MIC of MP-B was studied by qRT-PCR. It was found that MP-B had potent antimicrobial activity (MIC=1 μg/ml) associated with a 20-fold decrease in theadeB gene expression at the sub-MIC level. The stereochemical analysis using several automated servers confirmed that the AdeB protein is an inner membrane of the RND tripartite complex system with helix-turn-helix conformation and a pore rich in Phe, Ala, and Lys residue. Furthermore, 20 ligands were generated from the initial docked poses to create the correct protein-peptide complexes using the BioLiP pipeline. The pose showed high Z=1.2, C=1.41, TM=0.99, and RMSD=4.4 scores was selected for docking purposes. The molecular docking via AutoDock/Vina revealed that MP-B form H-bound with Val 499, Phe 454, Thr 474, Ser 461, Gly 465, and Tyr 468 residues of the AdeB helix-5 and caused a shift in the dihedral angle (Φ/Ψ) by distances of 9.0 Å, 9.3 Å, and 9.6 Å, respectively. This shift in folding was detected by AlphaFold 2 and influenced the overall druggability of the protein. From the above results, we concluded that MP-B can be a good candidate for bacterial efflux pump inhibition.
PATCRdb: Database of TCRs from data mining patent documents
Authors: Lee Yoona , Freitag Rebecca , Ganesan Rajkumar , Schwämmle Veit , Kumar Sandeep , Krawczyk Konrad
Published: January 2023

T-cells are crucial actuators of the innate immune system. Because their receptors recognize intracellular disease markers, there is considerable interest in developing them as novel biotherapies. Computational methods to support discovery, design and development of TCR-based therapeutics need robust repositories of curated sequence and structural information on TCRs. The urgency of this need is highlighted by the recent approval of the first TCR biotherapeutic, tebentafusp. In this work, we have collected patent data on TCR sequences to provide early access to TCRs that are in various stages of product and clinical development (pre-FDA approvals) and are already past the initial discovery / proof of concept (scientific publications) stages. We employ literature mining to identify patent documents disclosing TCR sequences. Such documents are further analyzed to provide a birds-eye view of TCR patenting landscape. We compile the information into a database available athttp://github.com/konradkrawczyk/patcrdbthat we hope should help TCR engineers.
Diagnosis of protozoa diarrhoea inCampylobacterpatients increases markedly with molecular techniques
Authors: Hayman David T S , Garcia-Ramirez Juan Carlos , Pita Anthony , Velathanthiri Niluka , Knox Matthew A , Ogbuigwe Paul , Baker Michael G , Rostami Kamran , Deroles-Main Jan , Gilpin Brent J.
Published: January 2023

Background

CryptosporidiumandGiardiaare major food and water-borne causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques for diagnostics. Here we investigate the level of protozoa coinfection identified by molecular methods inCampylobacterpositive samples missed through use of antigen-based assays and then investigated different molecular testing protocols.

Methods

We report the findings of two observational studies; the first among 111 people with diarrhoea during a largeCampylobacteroutbreak in Havelock North, and the second a study during normal surveillance activities among 158 people presenting with diarrhoea and a positiveCampylobactertest, but negativeCryptosporidiumand/orGiardiaantigen-based diagnostic test result. The molecular methods used for comparison with the antigen-based tests were in-house end-point PCR tests targeting thegp60gene forCryptosporidiumandgdhgene forGiardia. DNA extraction was performed with and without bead-beating and comparisons with commercial real-time quantitative (qPCR) were made using clinical samples diluted down to 10−5forCryptosporidiumpositive samples.

Results

The coinfection prevalence was 9% (n= 10, 3–15% 95%CI) forCryptosporidiumand 21% (n=23, 12– 29% 95%CI) forGiardiain the 111Campylobacterpatients of the Havelock North outbreak. The coinfection prevalence was 40% (n=62, 32-48% 95%CI) forCryptosporidiumand 1.3% (n=2, 0.2-4.5% 95%CI) forGiardiain the 158 routine surveillance samples. Sequencing identifiedCryptosporidium hominis, C. parvum, andGiardia intestinalisassemblages A and B among patients. We found no statistical difference in positive test results between samples using end-point PCR with or without bead-beating prior to DNA extraction, or between the in-house end-point PCR and qPCR. The qPCR Ct value was 36 (35-37 95%CI) for 1 oocyst, suggesting a high limit of detection.

Discussion

In surveillance and outbreak situations we found diagnostic serology testing substantially underdiagnosesCryptosporidiumandGiardiacoinfections inCampylobacterpatients. These findings suggest that the impact of protozoa infections may be underestimated, through underdiagnosis, but molecular techniques likely improve detection capabilities. Laboratories need to understand clinical, rather than analytical, test sensitivity, to allow clinicians to better understand the disease aetiologies of patients that enable better health advice.
Spatiotemporal and meteorological trends in dengue transmission in the Dominican Republic, 2015-2019
Authors: Robert Michael A. , Rodrigues Helena Sofia , Herrera Demian , de Mata Donado Campos Juan , Morilla Fernando , Águila Mejía Javier Del , Elena Guardado María , Skewes Ronald , Colomé-Hidalgo Manuel
Published: January 2023

Dengue has broadened its global distribution substantially in the past two decades, and many endemic areas are experiencing increases in incidence. The Dominican Republic recently experienced its two largest outbreaks to date with 16,836 reported cases in 2015 and 20,123 reported cases in 2019. With this upward trend in dengue transmission, developing tools to better prepare healthcare systems and mosquito control agencies is of critical importance. Before such tools can be developed, however, we must first better understand potential drivers of dengue transmission. To that end, we focus in this paper on determining trends between climate variables and dengue transmission with an emphasis on eight provinces and the capital city of the Dominican Republic in the period 2015-2019. We present summary statistics for dengue cases, temperature, precipitation, and relative humidity in this period, and we conduct an analysis of correlated lags between climate variables and dengue cases as well as correlated lags among dengue cases in each of the nine locations. We find that the southwestern province of Barahona had the largest dengue incidence in both 2015 and 2019. Among all climate variables considered, lags between temperature variables and dengue cases were the most highly correlated. We found that most locations had significant correlations at lags of zero weeks; however, both Barahona and the northern province of Monte Cristi had significantly correlated lags with other provinces at up to eight weeks. These results can be used to improve predictive models of dengue transmission in the country.
Visual field evaluation using Zippy Adaptive Threshold Algorithm (ZATA) Standard and ZATA Fast in patients with glaucoma and healthy individuals
Authors: Nasim Pinaz , Ramesh S Ve , Kuzhuppilly Neetha IR , Naik Preethi , Artes Paul H , Ballae Ganeshrao Shonraj
Published: January 2023

Purpose

To evaluate visual fields using Zippy Adaptive Thresholding Algorithm (ZATA) Standard and ZATA Fast among patients with glaucoma and healthy individuals.

Methods

We recruited 22 healthy participants and 55 patients with glaucoma from the ophthalmology clinics at Kasturba Hospital, Manipal, India. Inclusion criteria were age 35 to 85 years, best corrected visual acuity (BCVA) 0.3 logMAR or better. Glaucoma patients had characteristic glaucomatous optic disc damage. All participants were free from any other ocular pathology except from mild cataract. Participants performed ZATA Standard and ZATA Fast tests on a Henson 9000 perimeter and Swedish Interactive Thresholding Algorithm (SITA) Standard and SITA Fast tests on a Humphrey Field Analyser. Tests were repeated within 90 days to evaluate the test-retest variability.

Results

The mean difference in the mean deviation (MD) values of ZATA Standard and SITA Standard tests was 1.7 dB, and that in ZATA Fast and SITA Fast tests was 0.9 dB. The sensitivity values of ZATA and SITA tests differed by 3 dB. ZATA Standard and ZATA Fast were 30% and 6% faster than the corresponding SITA tests. Grayscale and probability maps varied slightly between the four tests but represented a similar visual field for most patients in the study.

Conclusions

ZATA Standard and ZATA Fast are suitable for clinical practice. However, differences between ZATA and SITA tests suggest that they should not be used interchangeably when monitoring over time.
Immunomorphogenesis in degenerative disc disease: the role of proinflammatory cytokines and angiogenesis factors
Authors: Pravdyuk N.G. , Novikova A.V. , Shostak N.A. , Buianova A.A. , Patsap O.I. , Raksha A.P. , Timofeyev V.T. , Feniksov V.M. , Nikolayev D.A. , Senko I.V.
Published: January 2023

Back pain (BP) due to degenerative spinal injury is a severe, often disabling disease and it tends to decrease age of onset. The cellular and molecular basis of the disc and adjacent structures degradation remains in the focus of pathophysiologists and pathomorphologists close attention. The aim of the study was to determine the expression level of proinflammatory cytokines (IL-1β, IL-6, IL-17) and angiogenesis markers (VEGF-A and CD31) in intervertebral disc (IVD) tissue, their association between each other and between disc degeneration in young people with discogenic BP. In patients who underwent discectomy for a disc herniation, a clinical examination, magnetic resonance imaging (MRI) of the lumbar spine, histological and immunohistochemical analyses of these factors in the disc tissue were performed in comparison with the parameters of healthy group samples (controls). Histology image analysis of IVD fragments of the degenerative disc disease (DDD) group detected zones of inflammatory infiltration, combined with vascularization, the presence of granulation tissue and clusters of chondrocytes in the tissue of nucleus pulposus (NP). Statistically significant expression of IL-1β, IL-6, IL-17 and VEGF-A (especially on the chondrocytes surface) and CD31 (in the disc matrix) was evident in the samples of the DDD group compared with the controls (p < 0.0001), that showed a strong correlation with the histological disc degeneration stage (r > 0.5; p < 0.0001). This denotes a high immunoinflammatory potential of chondrocytes and demonstrates their altered morphogenetic properties. The coincidence of the spatial expression of IL-1β and IL-17 in the perivascular zone endothelium and in the vascular lumen (p < 0.01) was found, which point at the inflammatory synergy of these cytokines. High expression of VEGF-A prevailed on the surface of chondrocytes in cell clusters compared to the matrix (p < 0.0001), indicating that the NP cells trigger the angiogenesis. The absence of CD31 in the cracks of NP with high expression in the disc matrix states the secondary nature of IVD defects due to degeneration, and not due to vascular ingrowth. A high number of patients with Modic changes according to MRI data shown the contribution of cytokines to the formation of reactive spondylitis and the clinical course of BP. These results obtained will help to development molecular/cellular targets and basic strategies for therapy of BP with DDD in the early stages in young people.
A single-cell atlas depicting the cellular and molecular features in human ligamental degeneration: a single cell combined spatial transcriptomics study
Authors: Yang Runze , Xu Tianhao , Zhang Lei , Ge Minghao , Yan Liwei , Li Jian , Fu Weili
Published: January 2023

Background

To systematically identify cell types in human ligament, investigate how ligamental cell identities, functions, and interactions participated the process of ligamental degeneration and explore the changes of ligamental microenvironment homeostasis in the disease progression.

Methods

Using single-cell RNA sequencing and spatial RNA sequencing of approximately 49356 cells, we created a comprehensive cell atlas of healthy and degenerated human anterior cruciate ligaments. We explored the variations of the cell subtypes’ spatial distributions and the different processes involved in the disease progression, linked them with ligamental degeneration process using computational analysis.

Results

We identified new fibroblast subgroups contributed to the disease and mapped out their spatial distribution in the tissue and revealed two stages of the degenerative process. We compared the cellular interactions between different tissue states and identified important signaling pathways may contribute to the disease.

Conclusion

This cell atlas provides the molecular foundation for investigating how ligamental cell identities, biochemical functions, and interactions contributed to the ligamental degeneration process. The discoveries revealed the pathogenesis of ligamental degeneration at single-cell and spatial level which is characterized by extracellular matrix remodeling. Our results provide new insights into the control of ligamental degeneration and potential clues to developing novel diagnostic and therapeutic strategies.

Funding

This study was funded by the National Natural Science Foundation of China (81972123, 82172508), Sichuan Science and Technology Program (2020YFH0075), Fundamental Research Funds for the Central Universities (2015SCU04A40), Chengdu Science and Technology Bureau Project (2019-YF05-00090-SN), and 1.3.5 Project for Disciplines of Excellence of West China Hospital Sichuan University (ZYJC21030, ZY2017301).
Using machine learning to design a short test from a full-length test of functional health literacy in adults - the development of a short form of the Danish TOFHLA
Authors: Hæsum Lisa Korsbakke Emtekær , Cichosz Simon Lebech , Hejlesen Ole Kristian
Published: January 2023

Introduction

Patients are compelled to become more involved in shared decision making with healthcare professionals in the self-management of chronic disease and general adherence to treatment. Therefore, it is valuable to be able to identify patients with low functional health literacy so they can be given special instructions about the management of chronic disease and medications. However, time spent by both patients and clinicians is a concern when introducing a screening instrument in the clinical setting, which raises the need for short instruments for assessing health literacy that can be used by patients without the involvement of healthcare personnel. This paper describes the development of a short version of the full-length Danish TOFHLA (DS-TOFHLA) that is easily applicable in the clinical context and where the use does not require a trained interviewer.

Materials and Methods

Data were collected as a part of a large-scale telehomecare project (TeleCare North), which was a randomized controlled trial that included 1225 patients with chronic obstructive pulmonary disease.

The DS-TOFHLA was developed solely using an algorithm-based selection of variables and multivariable linear regression models. A multivariable regression model was developed using an exhaustive search strategy.

Results

The exhaustive search showed that the number of items in the full-length TOFHLA could be reduced from 17 numeracy items and 50 reading comprehension items to 20 reading comprehension items while maintaining a correlation of r = 0.90. A generic model-based approach was developed, which is suitable for development of short versions of the TOFHLA in other languages, including the original American version.

Conclusions

This study demonstrated how a generic model-based approach could be applied in the development of a short version of the TOFHLA, thereby reducing the 67 items to 20 items in the short version. Furthermore, this study showed that the inclusion of numeracy items was not necessary. The development of the DS-TOFHLA presents an opportunity to reliably identify patients with inadequate functional health literacy in approximately 5 minutes without involvement of healthcare personnel. The approach may be used in the development of short versions of any scaling questionnaire.
Osseointegration of functionally-graded Ti6Al4V porous implants: Histology of the pore network
Authors: Deering Joseph , Mahmoud Dalia , Rier Elyse , Lin Yujing , do Nascimento Pereira Anna Cecilia , Titotto Silvia , Fang Qiyin , Wohl Gregory R. , Deng Feilong , Grandfield Kathryn , Elbestawi Mohamed A. , Chen Jianyu
Published: January 2023

The additive manufacturing of titanium into porous geometries offers a means to generate low-stiffness endosseous implants with a greater surface area to improve osseointegration. In order to optimize pore size in the scaffolds, it is important to first understand the timeline of osseointegration in pre-clinical models. In this work, selective laser melting was used to produce gyroid-based scaffolds with a uniform pore size of 300 μm or functionally-graded pore size from 600 μm to 300 μm before implantation in New Zealand white rabbit tibiae for 4 and 12 weeks. Initialin vitroassessment with Saos-2 cells showed favourable cell proliferation at pore sizes of 300 and 600 μm. At four weeks, histological observations indicated some residual inflammation alongside neovessel infiltration into the scaffold interior and some early apposition of mineralized bone tissue. At twelve weeks, both scaffolds were filled with a mixture of adipocyte-rich marrow, micro-capillaries, and mineralized bone tissue. X-ray microcomputed tomography showed a higher bone volume fraction (BV/TV) and percentage of bone-implant contact (BIC) in the implants with 300 μm pores than in the functionally-graded specimens, indicating that these smaller pore sizes may be favourable for osseointegration in leporine bone.

Graphical Abstract
Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics
Authors: Chang Alexis , Leutert Mario , Rodriguez-Mias Ricard A. , Villén Judit
Published: January 2023

Phosphotyrosine (pY) enrichment is critical for expanding fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY handles 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic and tyrosine specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.
ConDoR: Tumor phylogeny inference with a copy-number constrained mutation loss model
Authors: Sashittal Palash , Zhang Haochen , Iacobuzio-Donahue Christine A. , Raphael Benjamin J.
Published: January 2023

Tumors consist of subpopulations of cells that harbor distinct collections of somatic mutations. These mutations range in scale from single nucleotide variants (SNVs) to large-scale copy-number aberrations (CNAs). While many approaches infer tumor phylogenies using SNVs as phylogenetic markers, CNAs that overlap SNVs may lead to erroneous phylogenetic inference. Specifically, an SNV may be lost in a cell due to a deletion of the genomic segment containing the SNV. Unfortunately, no current single-cell DNA sequencing (scDNA-seq) technology produces accurate measurements of both SNVs and CNAs. For instance, recenttargetedscDNA-seq technologies, such as Mission Bio Tapestri, measure SNVs with high fidelity in individual cells, but yield much less reliable measurements of CNAs. We introduce a new evolutionary model, theconstrained k-Dollo model, that uses SNVs as phylogenetic markers and partial information about CNAs in the form of clustering of cells with similar copy-number profiles. This copy-number clustering constrains where loss of SNVs can occur in the phylogeny. We develop ConDoR (Constrained Dollo Reconstruction), an algorithm to infer tumor phylogenies from targeted scDNA-seq data using the constrainedk-Dollo model. We show that ConDoR outperforms existing methods on simulated data. We use ConDoR to analyze a new multi-region targeted scDNA-seq dataset of 2153 cells from a pancreatic ductal adenocarcinoma (PDAC) tumor and produce a more plausible phylogeny compared to existing methods that conforms to histological results for the tumor from a previous study. We also analyze a metastatic colorectal cancer dataset, deriving a more parsimonious phylogeny than previously published analyses and with a simpler monoclonal origin of metastasis compared to the original study.

Code availability

Software is available athttps://github.com/raphael-group/constrained-Dollo
The maternal microbiome regulates infant respiratory disease susceptibility via intestinal Flt3L expression and plasmacytoid dendritic cell hematopoiesis
Authors: Sikder Md. Al Amin , Rashid Ridwan B. , Ahmed Tufael , Sebina Ismail , Howard Daniel , Ullah Md. Ashik , Rahman Muhammed Mahfuzur , Lynch Jason P. , Curren Bodie , Werder Rhiannon B. , Simpson Jennifer , Bissell Alec , Morrison Mark , Walpole Carina , Radford Kristen J. , Kumar Vinod , Woodruff Trent M. , HuiYing Tan , Ali Ayesha , Kaiko Gerard E. , Upham John W. , Hoelzle Robert D. , Cuív Páraic Ó , Holt Patrick G. , Dennis Paul G. , Phipps Simon
Published: January 2023

Severe lower respiratory infection (sLRI) are a major cause of infant morbidity and mortality, and predispose to later chronic respiratory diseases such as asthma. Poor maternal diet during pregnancy is a risk factor for sLRI in the offspring. Here we demonstrate in mice that a maternal low-fibre diet (LFD) disrupts plasmacytoid and conventional dendritic cell (DC) hematopoiesis in the offspring, predisposing to sLRI and subsequent asthma. The LFD alters the composition of the maternal milk microbiome and assembling infant gut microbiome, ablating the induction of a developmental wave of the non-redundant DC growth factor Flt3L by neonatal intestinal epithelial cells. Therapy with a propionate-producing bacteria isolated from the milk of high-fibre diet-fed mothers, or supplementation with propionate, confers protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that regulates pDC hematopoiesis in early life and confers disease resistance.
Environmental constraints can explain clutch size differences between urban and forest blue tits: insights from an egg removal experiment
Authors: Pitt Mark D. , Capilla-Lasheras Pablo , Alhowiti Norah S.S. , Branston Claire J. , Carlon Eugenio , Boonekamp Jelle J. , Dominoni Davide M.
Published: January 2023

Urban environments present novel ecological challenges to wild species. Understanding whether species responses to urban living are adaptive or maladaptive is critical to predicting the impacts of urbanisation on biodiversity. In birds, urban populations generally exhibit reduced reproductive investment (clutch size) compared to forest populations. However, whether smaller clutches are adaptive, or a result of environmental constraints is unclear. Here, to investigate these two hypotheses, we quantified the ability of urban and forest blue tits (Cyanistes caeruleus) to lay new eggs upon egg removal. Consistent with the constraint hypothesis, our results suggest that urban females do not lay new eggs, at least to the same extent as forest birds. Meanwhile, forest birds laid approximately two additional eggs. As urban blue tits did not lay replacement eggs, our experiment resulted in a brood reduction and nestlings from urban experimental nests had higher survival than those from urban control nests, suggesting that females may be misjudging urban habitat quality and produce a clutch too large to be sustained. Taken together, our results suggest that urban females may experience constraints that limit egg formation and/or exacerbate the trade-off between female survival and egg production. This has important implications for urban green space management.
Rupture Strength of Living Cell Monolayers
Authors: Duque Julia , Bonfanti Alessandra , Fouchard Jonathan , Baldauf Lucia , Azenha Sara R. , Ferber Emma , Harris Andrew , Barriga Elias H. , Kabla Alexandre J. , Charras Guillaume
Published: January 2023

The ability of tissues to sustain mechanical stress and avoid rupture is a fundamental pillar of their function. Rupture in response to physiological levels of stress can be undesired, for example resulting from disease or genetic mutations, or be an integral part of developmental processes, such as during blastocoel formation in mouse or leg eversion in flies. Despite its importance, we know very little about rupture in cellularised tissues because it is a multi-scale phenomenon that necessitates comprehension of the interplay between mechanical forces and processes at the molecular and cellular scales. Using a combination of mechanical measurements, live imaging and computational modelling, we characterise rupture in epithelial monolayers. We show that, despite consisting of only a single layer of cells, monolayers can withstand surprisingly large deformations, often accommodating several-fold increases in their length before rupture. At large deformation, epithelia increase their stiffness multiple-fold in a process controlled by a supracellular network of keratin filaments. Perturbing keratin organisation fragilised monolayers and prevented strain stiffening. Using computational approaches, we show that, although the kinetics of adhesive bond rupture ultimately control tissue strength, tissue rheology and the history of deformation prior to failure set the strain and stress that the tissue reaches at the onset of fracture. Our data paint a picture of epithelia as versatile materials that combine resistance to shocks with deformability when subjected to low strain rates.
Subclonal somatic copy number alterations emerge and dominate in recurrent osteosarcoma
Authors: Kinnaman Michael D. , Zaccaria Simone , Makohon-Moore Alvin , Arnold Brian , Levine Max , Gundem Gunes , Arango Ossa Juan E. , Glodzik Dominik , Rodríguez-Sánchez M. Irene , Bouvier Nancy , Li Shanita , Stockfisch Emily , Dunigan Marisa , Cobbs Cassidy , Bhanot Umesh , You Daoqi , Mullen Katelyn , Melchor Jerry , Ortiz Michael V. , O’Donohue Tara , Slotkin Emily , Wexler Leonard H. , Dela Cruz Filemon S. , Hameed Meera , Glade Bender Julia L. , Tap William D. , Meyers Paul A. , Papaemmanuil Elli , Kung Andrew L. , Iacobuzio-Donahue Christine A
Published: January 2023

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses.MYCgain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such asCCNE1,RAD21,VEGFA, andIGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma.

Significance

Subclonal copy number clones emerged and dominated in relapsed osteosarcoma, withMYCgain/amplification being the defining characteristic in our cohort. Selective pressure from neoadjuvant chemotherapy revealed this clone at the time of primary resection, highlighting that genomic profiling at this time may identify clones that are selected for, or determine innate resistance to primary chemotherapy.
A Novel Protein for the Bioremediation of Gadolinium Waste
Authors: Lee Harvey D. , Grady Connor J. , Krell Katie , Strebeck Cooper , Good Nathan M. , Martinez-Gomez N. Cecilia , Gilad Assaf A.
Published: January 2023

Several hundreds of tons of gadolinium-based contrast agents (GBCAs) are being dumped into the environment every year. Although macrocyclic GBCAs exhibit superior stability compared to their linear counterparts, we have found that the structural integrity of chelates are susceptible to ultraviolet light, regardless of configuration. In this study, we present a synthetic protein termed GLamouR that binds and reports gadolinium in an intensiometric manner. We then explore the extraction of gadolinium from GBCA-spiked artificial urine samples and investigate if the low picomolar concentrations reported in gadolinium-contaminated water sources pose a barrier for bioremediation. Based on promising results, we anticipate GLamouR can be used for detecting and mining REEs beyond gadolinium as well and hope to expand the biological toolbox for such applications.
Persistent confined migration confers permanent nuclear and functional changes in migrating cells
Authors: Lope-Planelles Ana de , González-Novo Raquel , Madrazo Elena , Zamora-Carreras Héctor , Torrano Verónica , López-Menéndez Horacio , Roda-Navarro Pedro , Monroy Francisco , Redondo-Muñoz Javier
Published: January 2023

Nuclear deformability plays a critical role in cell migration. During this process, the remodeling of internal components of the nucleus has a direct impact on DNA damage and cell behavior; however, how persistent migration promotes nuclear changes leading to phenotypical and functional consequences remains poorly understood. Here, we described that the persistent migration through physical barriers was sufficient to promote permanent modifications in migratory-altered cells. We found that lamin B1 altered its localization, concomitant with morphological and transcriptional changes. Migratory-altered cells showed alterations in cellular functions such as DNA repair and cell migration. We applied biochemical and biophysical approaches to identify that confined conditions altered the biomechanical response of the nucleus. Mechanistically, we determined that actin dynamics controlled the redistribution of lamin, and the basal levels of DNA damage in migratory-altered cells. Our observations reveal a novel role for confined cell conditions in consistent nuclear and genomic alterations that might handle the genetic instability and cellular heterogeneity in aging diseases and cancer.

Highlights

Persistent confined migration promotes permanent mophological changes.

Lamin B1 is redistribution in the nucleus of migratory altered cells.

Migratory-altered cells exhibit transcriptional and functional changes related to cell migration and survival.

Actin polymerization controls nuclear changes induced by cell migration.
Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema
Authors: Lamaa A. , Creff J. , Benuzzi E. , Pujol f. , Draia-Nicolau T. , Nougué M. , Verdu L. , Morfoisse F. , Lacazette E. , Valet P. , Chaput B. , Gross F. , Gayon R. , Bouillé P. , Malloizel-Delaunay J. , Bura-Rivière A. , Prats A.C. , Garmy-Susini B.
Published: January 2023

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in lymphedematous arm compared to normal arm in patients. The role of APLN in LD was confirmed in APLN-knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of safe and non-integrative RNA-delivery LentiFlash®vector that will be evaluated for phase I/IIa clinical trial.
Transcriptome analysis reveals novel regulators of the Scots pine stilbene pathway
Authors: Paasela Tanja , Lim Kean-Jin , Pavicic Mirko , Harju Anni , Venäläinen Martti , Paulin Lars , Auvinen Petri , Kärkkäinen Katri , Teeri Teemu H.
Published: January 2023

Stilbenes are developmentally induced metabolites in Scots pine heartwood where they have important role in protecting wood from decaying fungi. The stilbene pathway is also stress inducible, and ultraviolet (UV)-C radiation was among the first discovered artificial stress activators of the pathway. Many conditions that activate the pathway are known, but the specific transcriptional regulators and the biosynthetic enzyme responsible for activating the stilbene precursor cinnamate in the pathway are unknown.

Here we describe the first large-scale transcriptomic analysis of pine needles in response to UV-C exposure and treatment with translational inhibitor cycloheximide, both activating the transcription of stilbene pathway genes, to uncover the candidates for the transcriptional regulation of the pathway and the cinnamate activating CoA ligase. We show that the regulation of pine stilbene pathway has shared features with grapevine stilbene pathway. In both species, the stilbene pathway was transcriptionally activated after UV-C treatment, protein phosphatase inhibitors and plant hormones ethylene and jasmonate.

The pine stilbene synthase promoter retains its inducibility when transformed in plants that normally do not synthesize stilbenes. Pine stilbene synthase promoter was able to activate the expression of a reporter gene in Arabidopsis in response to UV-C exposure and phosphatase inhibitors, but not as response to plant hormone treatment. This indicates that stilbene synthase gene regulation occurs both via ancient stress-response pathway(s) but also via species specific regulators. With transcriptomic approach we identified candidate enzymes for cinnamate acting CoA ligase and transcription factors regulating the pathway.
Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection
Authors: Bennett Allison K. , Richner Michelle , Mun Madeline D. , Richner Justin M.
Published: January 2023

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adult. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established anex vivoculture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population- and gene expression-level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.
Decomprolute: A benchmarking platform designed for multiomics-based tumor deconvolution
Authors: Feng Song , Calinawan Anna , Pugliese Pietro , Wang Pei , Ceccarelli Michele , Petralia Francesca , Gosline Sara JC
Published: January 2023

Tumor deconvolution is a reliable way to disentangle the diverse cell types that comprise solid tumors. To date, however, both the algorithms developed to deconvolve tumor samples, and the gold standard datasets used to assess the algorithms are geared toward the analysis of gene expression (e.g., RNA-seq) rather than protein levels in tumor cells. While gene expression is less expensive to measure, protein levels provide a more accurate view of immune markers. To facilitate the development as well as improve the reproducibility and reusability of multi-omic deconvolution algorithms, we introduce Decomprolute, a Common Workflow Language framework that leverages containerization to compare tumor deconvolution algorithms across multiomic data sets. Decomprolute incorporates the large-scale multiomic data sets produced by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), which include matched mRNA expression and proteomic data from thousands of tumors across multiple cancer types to build a fully open-source, containerized proteogenomic tumor deconvolution benchmarking platform. The platform consists of modular architecture and it comes with well-defined input and output formats at each module. As a result, it is robust and extendable easily with additional algorithms or analyses. The platform is available for access and use athttp://pnnl-compbio.github.io/decomprolute.

Motivation

To provide a comprehensive platform for algorithm developers and researchers to benchmark and run tumor deconvolution algorithms on multiomic data.
Durotaxis bridges phase transition as a function of tissue stiffnessin vivo
Authors: Zhu Min , Gu Bin , Thomas Evan , Tao Hirotaka , Yung Theodora M. , Zhang Kaiwen , Rossant Janet , Sun Yu , Hopyan Sevan
Published: January 2023

Physical processes ultimately drive morphogenetic cell movements. Two proposals are that 1) cells migrate toward stiffer tissue (durotaxis) and that 2) the extent of cell rearrangements reflects liquid-solid tissue phase. It is unclear whether and how these concepts are related. Here, we identify fibronectin-dependent tissue stiffness as a control variable that underlies and unifies these phenomenain vivo. In murine limb bud mesoderm, cells are either caged, move directionally by durotaxis or intercalate as a function of their location along a stiffness gradient. A unifying stiffness-phase transition model that is based on a Landau equation accurately predicts cell diffusivity upon loss or gain of fibronectin. Fibronectin is regulated by a WNT5A-YAP positive feedback pathway that controls cell movements, tissue shape and skeletal pattern. The results identify a key determinant of phase transition and show how durotaxis emerges in a mixed phase environmentin vivo.
High-yield, ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single molecule manipulation assays
Authors: Lin Yi-Yun , Brouns Tine , Kolbeck Pauline J. , Vanderlinden Willem , Lipfert Jan
Published: January 2023

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCR reactions: first ∼380 bp long DNA strands are generated that contain multiple labels, which are used as “megaprimers” in a second PCR reaction to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. We use DBCO-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under force, with an average lifetime of 60 ± 3 hours at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.
Heterogeneous pseudobulk simulation enables realistic benchmarking of cell-type deconvolution methods
Authors: Hu Mengying , Chikina Maria
Published: January 2023

Computational cell type deconvolution enables estimation of cell type abundance from bulk tissues and is important for understanding cell-cell interactions, especially in tumor tissues. With rapid development of deconvolution methods, many benchmarking studies have been published aiming for a comprehensive evaluation for these methods. Benchmarking studies rely on cell-type resolved single-cell RNA-seq data to create simulated pseudbulk datasets by adding individual cells-types in controlled proportions. In our work we show that the standard application of this approach, which uses randomly selected single cells, regardless of the intrinsic difference between them, generates synthetic bulk expression values that lack appropriate biological variance. We demonstrate why and how the current bulk simulation pipeline with random cells is unrealistic and propose a heterogeneous simulation strategy as a solution. Our heterogeneously simulated samples show realistic variance across hallmark gene-sets when comparing with real bulk samples from the TCGA dataset of the same tumor type. Using this new simulation pipeline to benchmark deconvolution methods we show that introducing biological heterogeneity has a notable effect on the results. Evaluating the robustness of different deconvolution approaches to heterogeneous simulation we find that reference-free methods that rely on simplex estimation perform poorly, marker-based methods and BayesPrism are most robust, while regress-based approaches fall in between. Importantly, we find that under the heterogeneous scenario marker based methods and BayesPrism outperform state of the art reference methods. Our findings highlight how different conceptual approaches can negate unmodeled heterogeneity and suggest that there is room for further methodological development.
A Transcriptional Cofactor Regulatory Network for theC. elegansIntestine
Authors: Horowitz Brent B. , Nanda Shivani , Walhout Albertha J.M.
Published: January 2023

Chromatin modifiers and transcriptional cofactors (collectively referred to as CFs) work with DNA-binding transcription factors (TFs) to regulate gene expression. In multicellular eukaryotes, distinct tissues each execute their own gene expression program for accurate differentiation and subsequent functionality. While the function of TFs in differential gene expression has been studied in detail in many systems, the contribution of CFs has remained less explored. Here we uncovered the contributions of CFs to gene regulation in theCaenorhabditis elegansintestine. We first annotated 366 CFs encoded by theC. elegansgenome and assembled a library of 335 RNAi clones. Using this library, we analyzed the effects of individually depleting these CFs on the expression of 19 fluorescent transcriptional reporters in the intestine and identified 216 regulatory interactions. We found that different CFs interact specifically with different promoters, and that both essential and intestinally expressed CFs exhibit the highest proportion of interactions. We did not find all members of CF complexes acting on the same set of reporters but instead found diversity in the promoter targets of each complex component. Finally, we found that previously identified activation mechanisms for theacdh-1promoter use different CFs and TFs. Overall, we demonstrate that CFs function specifically rather than ubiquitously at intestinal promoters and provide an RNAi resource for reverse genetic screens.