bluuebunny/crossref_metadata_2025 huggingface dataset:
Nation-wide mammography screening participation in Denmark during the COVID-19 pandemic: An observational study
Authors: Olesen Tina Bech , Jensen Henry , Møller Henrik , Jensen Jens Winther , Andersen Berit , Vejborg Ilse , Njor Sisse H.
Published: September 2022

Background

In most of the world, the mammography screening programmes were paused at the start of the pandemic, whilst mammography screening continued in Denmark. We examined the mammography screening participation during the COVID-19 pandemic in Denmark.

Methods

The study population comprised all women aged 50-69 years old invited to participate in mammography screening from 2016-2021 in Denmark based on data from the Danish Quality Database for Mammography Screening in combination with population-based registries. Using a generalised linear model, we estimated prevalence ratios (PR) and 95% confidence intervals (CI) of mammography screening participation within 90, 180 and 365 days since invitation during the pandemic in comparison with the previous years adjusting for age, year and month of invitation.

Results

The study comprised 1,828,791 invitations among 847,766 women. Before the pandemic, 80.2% of invitations resulted in participation in mammography screening within 90 days, 82.7% within 180 days and 83.1% within 365 days. At the start of the pandemic, the participation in screening within 90 days was reduced to 69.9% for those invited in pre-lockdown and to 76.5% for those invited in 1st lockdown. Extending the length of follow-up time to 365 days only a minor overall reduction was observed (PR=0.94; 95% CI: 0.93-0.95 in pre-lockdown and PR=0.97; 95% CI: 0.96-0.97 in 1st lockdown). A lower participation was; however, seen among immigrants and among women with a low income.

Conclusions

The short-term participation in mammography screening was reduced at the start of the pandemic, whilst only a minor reduction in the overall participation was observed with longer follow-up time indicating that women postponed screening. Some groups of women; nonetheless, had a lower participation indicating that the social inequity in screening participation was exacerbated during the pandemic.
Vasoactive Endothelial Growth Factor and Heat Shock Protein Gene Expression Response in Kawasaki Disease
Authors: Rashid Asrar , Alkhzaimi Hoda , Benakatti Govind , Malik Zainab A. , Sharma Varun , Sharma Anuka , Malik Rayaz , Quraishi Nasir , Shaikh Guftar , Al-Dubai Ahmed , Hussain Amir
Published: September 2022

Kawasaki Disease (KD) is a childhood vasculitis primarily affecting medium-sized arteries, which can lead to severe complications, particularly with respect to coronary artery disease (CAD). The impact of thermal stress on KD coronary artery pathogenesis, in association with prolonged fever and inflammation, remains unclear. In this study, we hypothesized that altered gene expression (GE) of angiogenesis-inducing Heat Shock Proteins (HSPs) is associated with KD-CAD through pro-inflammation. Transcriptomic analysis was performed using the three largest KD peripheral blood studies in the clinical literature (KD1-KD3), and one study direct from coronary artery tissue (KD4). The analysis revealed a significant increase in TNF and NFKB1 GE, indicating the presence of inflammation based on gene expression profiles. Gene set enrichment analysis (GSEA) of KD1-KD3 datasets identified inflammatory pathways, including TNFA signaling via NFKB, IL6 JAK STAT 3 Signalling, and p53 (Heat Shock Protein 90). The study also focused on specific HSPs known to be associated with angiogenesis, namely HSPB1, HSPA1A, and HSP90AB1. The temporal transcript model (TTM) consistently showed up-regulation of pro-inflammatory genes VEGF-A, TNF, and NFKB1, as well as up-regulation of HSPA1A. GSEA revealed gene ontology pathways associated with VEGF production. These findings suggest that the binding of VEGF-A or VEGF-B to their receptors could potentially impact the coronary artery in KD. Additionally, the up-regulation of the gene HSPAB1 in KD has not been described previously. In contrast, KD4 showed no differential GE for the studied genes potentially related to end-stage KD. This study provides valuable insights into VEGF and HSPs in KD-associated inflammation. Future research should focus on developing a VEGF-HSP CAD model to explore implications for KD biomarking as well as developing precision management strategies.
Lysosome repair by ER-mediated cholesterol transfer
Authors: Radulovic Maja , Wenzel Eva Maria , Gilani Sania , Holland Lya K.K. , Lystad Alf Håkon , Phuyal Santosh , Olkkonen Vesa M. , Brech Andreas , Jäättelä Marja , Maeda Kenji , Raiborg Camilla , Stenmark Harald
Published: September 2022

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by formation of contacts with the endoplasmic reticulum (ER), which depend on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.
Serum amyloid alpha 1-2 are not required for systemic inflammation in the 4T1 murine breast cancer model
Authors: He Chenfeng , Konishi Riyo , Harata Ayano , Nakamura Yuki , Mizuno Rin , Yoda Mayuko , Toi Masakazu , Kawaguchi Kosuke , Kawaoka Shinpei
Published: September 2022

Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause systemic inflammation. Despite the well-known coincidence of acute phase response and systemic inflammation, the direct roles of SAA proteins in systemic inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in systemic inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver and bone marrow. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. This study clarifies the negligible contribution of SAA1-2 proteins in systemic inflammation in the 4T1 breast cancer model.
Biochemical identification of a nuclear coactivator protein required for AtrR-dependent gene regulation in Aspergillus fumigatus
Authors: Paul Sanjoy , Ror Shivani , McDonald W. Hayes , Moye-Rowley W. Scott
Published: September 2022

Azole drugs represent the primary means of treating infections associated with the filamentous fungal pathogen Aspergillus fumigatus. A central player in azole resistance is the Zn2Cys6 zinc cluster-containing transcription factor AtrR. This factor stimulates expression of the both the cyp51A gene encoding the azole drug target enzyme as well as an ATP-binding cassette transporter-encoding gene called abcG1 (aka cdr1B). We have used a fusion protein between AtrR and the tandem affinity purification (TAP) moiety to purify proteins that associate with AtrR from A. fumigatus. Protein fractions associated with AtrR-TAP were subjected to MudPIT mass spectrometry and one of the proteins identified was encoded by the AFUA_6g08010 gene. We have designated this protein NcaA (Nuclear Co-Activator of AtrR). Loss of ncaA caused a reduction in voriconazole resistance and drug-induced abcG1 expression, although did not impact induction of cyp51A transcription. We confirmed association of AtrR and NcaA by co-immunoprecipitation from otherwise wild-type cells. Expression of fusion proteins between AtrR and NcaA with green fluorescent protein allowed determination that these two proteins were localized in the A. fumigatus nucleus. Together, these data support the view that NcaA is required for nuclear gene transcription controlled by AtrR.

Importance

Aspergillus fumigatus is the major filamentous fungal pathogen in humans and is susceptible to the azole antifungal class of drugs. However, loss of azole susceptibility has been detected with increasing frequency in the clinic and infections associated with these azole resistant isolates linked to treatment failure and worse outcomes. Many of these azole resistant mutant strains contain mutant alleles of the cyp51A gene encoding the azole drug target. A transcription factor essential for cyp51A gene transcription has been identified and designated AtrR. AtrR is required for azole inducible cyp51A transcription but we know little of the regulation of this transcription factor. Using a biochemical approach, we identify a new protein called NcaA that is involved in regulation of AtrR at certain target gene promoters. Understanding the mechanisms controlling AtrR function is an important goal in preventing or reversing azole resistance in this pathogen.
Influence of high-energy laser therapy to the patellar tendon on its ligamentous microcirculation: a quasi-experiment
Authors: Brandl Andreas , Egner Christoph , Reisser Ursel , Lingenfelder Christian , Schleip Robert
Published: September 2022

Laser therapeutic applications, such as the use of high energy lasers (HILT), are widely used in physical therapy, but basic studies on the mechanisms of action of HILT on tendinous/ligamentous tissue are largely lacking. The aim of this study was to investigate microcirculatory changes of the patellar tendon by HILT. 21 healthy volunteers were treated with HILT. Before and after the intervention, as well as 10 minutes later, the microcirculation was measured by noninvasive laser spectroscopy (O2C device). Tissue temperature was recorded at the measurement time points using thermography. Blood flow increased significantly by 86.38 AU (p < 0.001) after the intervention and by 25.76 AU (p < 0.001) at follow-up. Oxygen saturation increased by 20.14% (p < 0.001) and 13.48%, respectively (p < 0.001), whereas relative hemoglobin decreased by 6.67 AU and 7.90 AU, respectively. Tendon temperature increased by 9.45° and 1.94° Celsius, respectively. Acceleration of blood flow by improving the flow properties of erythrocytes and platelets may have caused the results.
Towards optimised extracellular vesicle proteomics from cerebrospinal fluid
Authors: Kangas Petra , Nyman Tuula A. , Metsähonkala Liisa , Burns Cameron , Tempest Robert , Williams Tim , Karttunen Jenni , Jokinen Tarja S.
Published: September 2022

The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean ± standard deviation. Using proteomics, SEC fractions 3-5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4-5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6ml, 3ml, 1ml, and 0.5ml) to evaluate the effect on the proteomic profile. Even with a 0.5ml starting volume, 743±77 or 345±88 proteins were identified depending on whether ‘matches between runs’ was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5ml of canine CSF.
Highly Sensitive and Specific Amyloid-β Oligomers Detection Using CRISPR-Cas12a Probe System aided by Graphene Oxide for Early Diagnosis of Alzheimer’s Diseases
Authors: Tang Jiaqi , Zhu Zongwei , Yang Chuanxu
Published: September 2022

Alzheimer’s disease (AD) is highly age-specific that results in cognitive incapability including dementia. It is proposed that AD is caused by the dysfunctional gene producing excessive amyloid-β protein that eventually results in aggregated form of amyloid-β Oligomers (AβO). We couple the CRISPR-Cas12a system with a specially designed single-stranded DNA (ssDNA) aptamer that specifically forms a hybrid complex with AβO. We exploit the aptamer as the substrate for the crRNA-aptamer duplex due to its high flexibility, sensitivity, and selectivity. We further adopted the graphene oxide (GO) fluorescent system to couple with our CRISPR-Cas12a probe system. Our coupled probe system can effectively detect the presence of AβO by quantitatively returning fluorescent signals with high specificity and accuracy.
Integrative Single Cell Multiomic Profiling Analysis Reveals HOX-PBX Gene Regulatory Network Contributing to the Survival of mTOR Hyperactive Cells
Authors: Olatoke Tasnim F. , Wagner Andrew , Astrinidis Aristotelis A. , Guo Minzhe , Zhang Erik Y. , Zhang Alan G. , Mattam Ushodaya , Chilton Kyla , Kopras Elizabeth J. , Gupta Nishant , Smith Eric P. , Karbowniczek Magdalena , Markiewkski Maciej M. , Wikenheiser-Brokamp Kathryn , Whitsett Jeffrey A. , McCormack Francis X. , Xu Yan , Yu Jane J.
Published: September 2022

Lymphangioleiomyomatosis (LAM) is a rare, debilitating lung disease that predominantly affects women of reproductive age. LAM is characterized by the infiltration of the lungs by abnormally proliferating smooth muscle-like cells of unknown origin via an estrogen-dependent metastatic mechanism. LAM cells carry deleterious mutations of tuberous sclerosis complex (TSC1/TSC2) genes, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) and ultimately dysregulated cell growth. Sirolimus, an FDA approved mTORC1 inhibitor and current best-choice medication for LAM stabilizes lung function in most LAM patients. However, it requires sustained application and remains inefficacious in some patients. The greatest barriers to finding a cure for LAM include its undetermined origin and unclear underlying pathogenesis. Our study aims to advance knowledge on the origin of LAM, and ultimately serve as a premise for the development of novel therapeutic targets for LAM. Single cell RNA sequencing (scRNA-seq) is a powerful tool in biomedical research that informs gene expression differences at the cellular level and may provide insights into the most fundamental origin of LAM cells. Our scRNA-seq analysis of LAM cells revealed a unique population of cells (LAMCORE), expressing uterine-similar homeobox transcription factors (HOX) and Pre-B-cell leukemia homeobox 1 (PBX1), which are absent in normal lung, suggesting that the uterus is the primary origin of LAM. PBX1 is a transcription factor critical for female reproductive tract development and maintenance, and its overexpression is implicated in some female reproductive cancers. In this study we hypothesize that PBX1 promotes survival and lung colonization of LAM (TSC2-null) cells. Using LAM patient-derived cells, we validated the transcriptional profile, gene expression and protein levels of PBX1. We have the first functional evidence that PBX1 and its downstream targets are upregulated in LAM cells. In a mouse model of LAM, we monitored the effect of suppression of PBX1 by short hairpin RNA-mediated gene silencing on lung colonization and tumor growth. We also found that pharmacological suppression of PBX1 attenuates LAM lung colonization and promotes death of LAM cells in vivo and vitro. Our data collectively suggests that PBX1 is a critical regulator of LAM progression.
Spatially Resolved Single-cell Translatomics at Molecular Resolution
Authors: Zeng Hu , Huang Jiahao , Ren Jingyi , Wang Connie Kangni , Tang Zefang , Zhou Yiming , Aditham Abhishek , Shi Hailing , Sui Xin , Wang Xiao
Published: September 2022

The precise control of mRNA translation is a crucial step in post-transcriptional gene regulation of cellular physiology. However, it remains a major challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolutions. Here, we report the development of RIBOmap, a three-dimensional (3D) in situ profiling method to detect mRNA translation for thousands of genes simultaneously in intact cells and tissues. By applying RIBOmap to 981 genes in HeLa cells, we revealed remarkable dependency of translation on cell-cycle stages and subcellular localization. Furthermore, we profiled single-cell translatomes of 5,413 genes in the adult mouse brain tissue with a spatial cell atlas of 62,753 cells. This spatial translatome profiling detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks. Together, RIBOmap presents the first spatially resolved single-cell translatomics technology, accelerating our understanding of protein synthesis in the context of subcellular architecture, cell types, and tissue anatomy.
The Rho GTPase exchange factor Vav2 promotes extensive age-dependent rewiring of the skin stem cell transcriptome
Authors: Lorenzo-Martín L. Francisco , Bustelo Xosé R.
Published: September 2022

Both the number and regenerative activity of skin stem cells (SSCs) are regulated by Vav2, a GDP/GTP exchange factor involved in the catalytic stimulation of the GTPases Rac1 and RhoA. However, whether Vav2 signaling changes in SSCs over the mouse lifespan is not yet known. Using a mouse knock-in mouse model, we now show that the expression of a catalytically-active version of Vav2 (Vav2Onc) promotes an extensive rewiring of the overall transcriptome of SSCs, the generation of new transcription factor hubs, and the synchronization of many transcriptional programs associated with specific SSC states and well-defined signaling pathways. Interestingly, this transcriptome rewiring is not fixed in time, as it involves the induction of 15 gene expression waves with diverse distribution patterns during the life of the animals. These expression waves are consistent with the promotion by Vav2Onc of several functional SSC states that differ from those normally observed in wild-type SSCs. These results further underscore the role of Vav2 in the regulation of the functional state of SSCs. They also indicate that, unlike other Vav2-dependent biological processes, the signaling output of this exchange factor is highly contingent on age-dependent intrinsic and/or extrinsic SSC factors that shape the final biological readouts triggered in this cell type.

AUTHOR SUMMARY

Skin stem cells (SSCs) are essential for the homeostatic balance of the skin, yet little is known to date about the biological and molecular mechanisms that modulate their abundance, long-term stability, or functional states during ageing. To address this issue, in this work we have used a genetically-engineered gain-of-function mouse model for Vav2, a Rho guanine nucleotide exchange factor (GEF) that has been recently shown to be involved in skin stem cell homeostasis. By performing time-course genome-wide expression analyses combined with a number of computational methods, here we show that: (i) Vav2 plays a critical role in regulating the functional state of SSCs, and (ii) the signaling output of constitutively active Vav2 is highly contingent on age-dependent intrinsic and/or extrinsic SSC factors that shape the final biological readouts triggered in this cell type. We believe that these data represent, to our knowledge, one of the first examples of the time-dependent output of an oncogenic version of a Rho GEF along a wide time interval in mice.
Cannabidiol targets a modulatory system for excitatory-inhibitory synaptic coordination, contributing to its anti-seizure action
Authors: Rosenberg Evan , Chamberland Simon , Bazelot Michael , Nebet Erica R. , Wang Xiaohan , McKenzie Sam , Jain Swati , Greenhill Stuart , Wilson Max , Salah Alejandro , Bailey Shanice , Patra Pabitra Hriday , Rose Rebecca , Chenouard Nicolas , Sun Simon D. , Jones Drew , Buzsáki György , Devinsky Orrin , Woodhall Gavin , Scharfman Helen , Whalley Benjamin , Tsien Richard
Published: September 2022

Cannabidiol (CBD), a non-euphoric component of cannabis, reduces seizures in multiple forms of pediatric epilepsy, but the mechanism(s) of anti-seizure action remain unclear. In one leading model, CBD acts at glutamatergic axon terminals, blocking pro-excitatory actions of an endogenous membrane phospholipid, lysophosphatidylinositol (LPI), at the G protein-coupled receptor GPR55. However, the impact of LPI-GPR55 signaling at inhibitory synapses and in epileptogenesis remains underexplored. We found that LPI transiently increased hippocampal CA3→CA1 excitatory presynaptic release probability and evoked synaptic strength in WT mice, while attenuating inhibitory postsynaptic strength by decreasing GABAARγ2 and gephyrin puncta. Effects of LPI at both excitatory and inhibitory synapses were eliminated by CBD pretreatment and absent after GPR55 deletion. Acute pentylenetrazole-induced seizures elevated levels of GPR55 and LPI, and chronic lithium pilocarpine-induced epileptogenesis potentiated the pro-excitatory effects of LPI. We propose that CBD exerts potential therapeutic effect both by blocking synaptic effects of LPI and dampening hyperexcitability.
Genetic diversity, paternal origin and pathogen resistance inCataglyphisdesert ants
Authors: Eyer Pierre-André , Guery Pierre-Antoine , Aron Serge
Published: September 2022

Group diversity is usually associated with a reduced risk of disease outbreak and a slower rate of pathogen transmission. In social insects, multiple mating by queens (polyandry) evolved several times although reducing worker’s inclusive fitness. One major hypothesis suggests that polyandry has been selected for to mitigate the risk of outbreak thanks to increased genetic diversity within colonies. We investigated this hypothesis in the antCataglyphis mauritanica, in which nestmate workers are produced by several clonal, single-mated queens. Using natural colonies, we correlated genetic diversity with worker survival to a fungal entomopathogen. We further tested whether workers from different paternal lineages (but a common maternal genome) show differential resistance in experimentally singleor multiple-patriline groups, and whether an increased number of patrilines in a group improved disease incidence.

We show that workers from distinct patrilines vary in their resistance to pathogen in single-patriline colonies, but the difference among patrilines disappears when they are mixed in multiple-patriline colonies. Furthermore, pathogen resistance was affected by the number of patrilines in a group, with twoand three-patriline groups being more resistant than single-patriline groups. However, resistance did not differ between groups made of two and three patrilines; similarly, it was not associated with genetic diversity in natural colonies. Overall, our results suggest that collective disease defenses might homogenize workers’ resistance from different patrilines and, thereby, stabilize colony resistance.
Interactions between insect vectors and plant pathogens span the parasitism-mutualism continuum
Authors: Santiago Ma. Francesca M. , King Kayla C. , Drew Georgia C.
Published: September 2022

Plants infected with vector-borne pathogens can suffer severe negative consequences, but the extent to which phytopathogens affect the fitness of their vector hosts remains unclear. Evolutionary theory predicts that selection on vector-borne pathogens should favour low virulence or mutualistic phenotypes in the vector, traits facilitating effective transmission between plant hosts. Here, we use a multivariate meta-analytic approach on 115 effect sizes across 34 unique plant-vector-pathogen systems to quantify the overall effect of phytopathogens on vector host fitness. In support of theoretical models, we report that phytopathogens overall have a neutral fitness effect on vector hosts. However, the range of possible fitness outcomes are diverse and span the parasitism-mutualism continuum. Contrary to previous predictions we found no evidence that transmission strategy, or the direct effects and indirect (plant-mediated) effects, of phytopathogens have divergent fitness outcomes for the vector. We discuss these findings in the context of plant – pathogen – vector ecology.
The Divergence of Mean Phenotypes Under Persistent Directional Selection
Authors: Devi Archana , Speyer Gil , Lynch Michael
Published: September 2022

Numerous organismal traits, particularly at the cellular level, are likely to be under persistent directional selection across phylogenetic lineages. For such traits, unless all mutations affecting such traits have large enough effects to be efficiently selected in all species, gradients in mean phenotypes are expected to arise as a consequence of differences in the power of random genetic drift, which varies by approximately five orders of magnitude across the Tree of Life. Prior theoretical work examining the conditions under which such gradients can arise focused on the simple situation in which all genomic sites affecting the trait have identical and constant mutational effects. Here, we extend this theory to incorporate the more biologically realistic situation in which mutational effects on a trait differ among nucleotide sites. Pursuit of such modifications lead to the development of semi-analytic expressions for the ways in which selective interference arises via linkage effects in single-effect models, which then extend to more complex scenarios. The theory developed clarifies the conditions under which mutations of different selective effects mutually interfere with each others’ fixation, and shows how variance in effects among sites can substantially modify and extend the expected scaling relationships between mean phenotypes and effective population sizes.
FAM237A, rather than peptide PEN and proCCK56-63, is a ligand of the orphan receptor GPR83
Authors: Li Hao-Zheng , Wang Ya-Fen , Shao Xiao-Xia , Liu Ya-Li , Xu Zeng-Guang , Wang Shi-Long , Guo Zhan-Yun
Published: September 2022

G protein-coupled receptor 83 (GPR83) is primarily expressed in the brain and is implicated in the regulation of energy metabolism and some behaviors. Recently, the PCSK1N/proSAAS-derived peptide PEN, the procholecystokinin-derived peptide proCCK56-63, and family with sequence similarity 237 member A (FAM237A) were all reported as agonists of GPR83. However, these results have not yet been reproduced by other laboratories and thus GPR83 is still officially an orphan receptor. The PEN and proCCK56-63 share sequence similarity; however, they are completely different from FAM237A, raising doubts that all of them are ligands of GPR83. To identify its actual ligand(s), in the present study we developed a NanoLuc Binary Technology (NanoBiT)-based ligand-binding assay, fluorescent ligand-based visualization, and a NanoBiT-based β-arrestin recruitment assay for human GPR83. Using these assays, we demonstrated that mature human FAM237A could bind to GPR83 with nanomolar range affinity, which activated this receptor and induced its internalization in transfected human embryonic kidney 293T cells. However, we did not detect any interaction of PEN and proCCK56-63 with GPR83 using these assays. Thus, the results confirmed that FAM237A is an agonist of GPR83, but did not support PEN and proCCK56-63 as ligands of this receptor. Clarification of its actual endogenous agonist will pave the way for further functional studies of this brain-specific receptor. The present study also provided an efficient approach for the preparation of mature FAM237A, which would facilitate further functional studies of this difficult-to-make peptide in the future.
CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer
Authors: Houthuijzen Julia M. , de Bruijn Roebi , van der Burg Eline , Drenth Anne Paulien , Wientjens Ellen , Filipovic Tamara , Bullock Esme , Brambillasca Chiara S. , Nieuwland Marja , de Rink Iris , van Diepen Frank , Klarenbeek Sjoerd , Kerkhoven Ron , Brunton Valerie G. , Scheele Colinda L.G.J. , Boelens Mirjam C. , Jonkers Jos
Published: September 2022

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice we determined that CAFs in both invasive lobular breast cancer and triple negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo tracing and in vitro studies revealed the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. In vitro functional assays showed that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data show that CD26+ and CD26- NFs transform into distinct CAF subpopulations in breast cancer.
Adjusting for genetic confounders in transcriptome-wide association studies leads to reliable detection of causal genes
Authors: Zhao Siming , Crouse Wesley , Qian Sheng , Luo Kaixuan , Stephens Matthew , He Xin
Published: September 2022

Expression Quantitative Trait Loci (eQTLs), provide valuable information on the effects of genetic variants. Many methods have been developed to leverage eQTLs to nominate candidate genes of complex traits, including colocalization analysis, transcriptome-wide association studies (TWAS), and Mendelian Randomization (MR)-based methods. All these methods, however, suffer from a key problem: when using the eQTLs of a gene to assess its role in a trait, nearby variants and nearby genetic components of expression of other genes can be correlated with the eQTLs of the test gene, while affecting the trait directly. These “genetic confounders” often lead to false discoveries. We introduced a novel statistical framework to address this challenge. Our method, causal-TWAS (cTWAS), borrowed ideas from statistical fine-mapping, and allowed us to adjust all genetic confounders. In our simulations, we found that existing methods based on TWAS, colocalization or MR all suffered from high false positive rates, often greater than 50%. In contrast, cTWAS showed calibrated false positive rates while maintaining power. Application of cTWAS on several common traits highlighted the weakness of existing methods and discovered novel candidate genes. In conclusion, cTWAS is a novel statistical framework to integrate eQTL and GWAS data, enabling reliable gene discoveries.
Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA): a live-imaging method for detecting epithelial barrier breaches with spatiotemporal precision
Authors: Higashi Tomohito , Stephenson Rachel E. , Schwayer Cornelia , Huljev Karla , Heisenberg Carl-Philipp , Chiba Hideki , Miller Ann L.
Published: September 2022

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance (TER), which measures the ion flux across epithelia, or by adding traceable macromolecules to one side of the epithelium and monitoring their passage to the other side. While these methods effectively measure changes to global barrier function, they are not sensitive enough to detect local or transient disruptions in the barrier, and they do not reveal the location of barrier breaches within the context of cell or tissue morphology. Therefore, we developed a method that we named Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction (TJ) leaks with high spatial and temporal resolution (Stephenson et al., 2019; Varadarajan et al., 2021). Here, we present expanded applications for ZnUMBA. First, we show that ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin dynamics following laser injury of the junction. We also demonstrate that ZnUMBA can be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby Canine Kidney II (MDCK II) epithelial cells. ZnUMBA is a powerful and flexible method that, with some optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.
Down the Penrose stairs: How selection for fewer recombination hotspots maintains their existence
Authors: Baker Zachary , Przeworski Molly , Sella Guy
Published: September 2022

In many species, meiotic recombination events tend to occur in narrow intervals of the genome, known as hotspots. In humans and mice, double strand break (DSB) hotspot locations are determined by the DNA-binding specificity of the zinc finger array of the PRDM9 protein, which is rapidly evolving at residues in contact with DNA. Previous models explained this rapid evolution in terms of the need to restore PRDM9 binding sites lost to gene conversion over time, under the assumption that more PRDM9 binding always leads to more DSBs. In recent experimental work, however, it has become apparent that PRDM9 binding on both homologs facilitates DSB repair, and moreover that, in the absence of enough symmetric binding, meiosis no longer progresses reliably. We therefore consider the possibility that the benefit of PRDM9 stems from its role in coupling DSB formation and efficient repair. To this end, we model the evolution of PRDM9 from first principles: from its binding dynamics to the population processes that govern the evolution of the zinc finger array and its binding sites in the genome. As we show, the loss of a small number of strong binding sites leads to the use of a greater number of weaker ones, resulting in a sharp reduction in symmetric binding and favoring new PRDM9 alleles that restore the use of a smaller set of strong binding sites. This decrease in PRDM9 binding symmetry and in its ability to promote DSB repair drive the rapid zinc finger turnover. These results imply that the advantage of new PRDM9 alleles is in limiting the number of binding sites used effectively, rather than in increasing net PRDM9 binding, as previously believed. By extension, our model suggests that the evolutionary advantage of hotspots may have been to increase the efficiency of DSB repair and/or homolog pairing.
PINK1 supports colorectal cancer growth by regulating the labile iron pool
Authors: Chen Brandon , Das Nupur K. , Talukdar Indrani , Singhal Rashi , Castillo Cristina , Andren Anthony , Mancias Joseph D. , Lyssiotis Costas A. , Shah Yatrik M.
Published: September 2022

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. We show that PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth following nutrient stress. However, the mechanism by which PINK1 maintains colon cancer growth remains equivocal. Inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited colon cancer cell proliferation, whereas disruption of other mitophagy receptors did not similarly impact cellular proliferation. Mechanistically, we observed a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione following PINK1 KD. Mitochondria are important hubs for storing iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. An increase iron storage protein ferritin and a decrease labile iron pool was observed in PINK1 KD cells. However, neither total cellular iron nor markers of iron starvation/overload were affected. Cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). Overexpressing nuclear receptor coactivator 4 (NCOA4), a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. We demonstrate that PINK1 regulates intracellular iron availability by integrating mitophagy to ferritinophagy. In conclusion, these results indicate that PINK1 is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells.
Phylogenetic inference from single-cell RNA-seq data
Authors: Liu Xuan , Griffiths Jason , Bishara Isaac , Liu Jiayi , Bild Andrea H. , Chang Jeffrey T.
Published: September 2022

Tumors are comprised of subpopulations of cancer cells that harbor distinct genetic profiles and phenotypes that evolve over time and during treatment. By reconstructing the course of cancer evolution, we can understand the acquisition of the malignant properties that drive tumor progression. Unfortunately, recovering the evolutionary relationship of individual cancer cells linked to their phenotypes remains a difficult challenge. To address this issue, we have developed PhylinSic, a method that reconstructs the phylogenetic relationships among cells linked to their gene expression profiles from single cell RNA-sequencing (scRNA-Seq) data, and showed that it was robust to the low read depth, drop-out, and noisiness of scRNA-Seq data. This method called nucleotide bases from scRNA-Seq reads using a probabilistic smoothing approach, and then estimated a phylogenetic tree using a Bayesian modeling algorithm. We evaluated PhylinSic and showed that it identified evolutionary relationships resulting from selective events such as drug selection and metastasis and was sensitive enough to identify subclones from genetic drift. Finally, we applied methods of phylogenetic inference and found that breast tumors resistant to chemotherapies harbored two genetic lineages that independently manifested high predicted activity of K-Ras and β-catenin, potentially acquired by distinct mechanisms through convergent evolution. This suggested that therapeutic strategies may need to target multiple lineages to be durable. Taken together, these results demonstrated that PhylinSic provides a framework to model the evolution and link the genotypes and phenotypes of cells within a tumor or cohort of monophyletic tumors using scRNA-Seq.
Diet-induced obesity alters intestinal monocyte-derived and tissue-resident macrophages in female mice independent of TNF
Authors: Breznik Jessica A. , Jury Jennifer , Verdú Elena F. , Sloboda Deborah M. , Bowdish Dawn M. E.
Published: September 2022

Macrophages are essential for homeostatic maintenance of the anti-inflammatory and tolerogenic intestinal environment, yet monocyte-derived macrophages can promote local inflammation. Pro-inflammatory macrophage accumulation within the intestines may contribute to the development of systemic chronic inflammation and immunometabolic dysfunction in obesity. Using a model of high fat diet-induced obesity in C57BL/6J female mice, we assessed intestinal permeability by in vitro and in vivo assays, and quantitated intestinal macrophages in ileum and colon tissues by multicolour flow cytometry after short (6 weeks), intermediate (12 weeks), and prolonged (18 weeks) diet allocation. We characterized monocyte-derived CD4−TIM4− and CD4+TIM4− macrophages, as well as tissue-resident CD4+TIM4+ macrophages. Diet-induced obesity had tissue and time-dependent effects on intestinal permeability, as well as monocyte and macrophage numbers, surface phenotype, and intracellular production of the cytokines IL-10 and TNF. We found that obese mice had increased paracellular permeability, in particular within the ileum, but this did not elicit recruitment of monocytes, nor a local pro-inflammatory response by monocyte-derived or tissue-resident macrophages, in either the ileum or colon. Proliferation of monocyte-derived and tissue-resident macrophages was also unchanged. Wildtype and TNF−/- littermate mice had similar intestinal permeability and macrophage population characteristics in response to diet-induced obesity. These data are unique from reported effects of diet-induced obesity on macrophages in metabolic tissues, as well as outcomes of acute inflammation within the intestines, and collectively indicate that TNF does not mediate effects of diet-induced obesity on intestinal monocyte-derived and tissue-resident intestinal macrophages in young female mice.

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Acclimating to degraded environments: The social rationale for swift action on restoration
Authors: Karatayev Vadim A. , Wilson Robyn S. , Webster D.G. , Axelrod Mark , Bauch Chris T. , Anand Madhur
Published: September 2022

As environmental degradation progresses, economies and societies adapt to the loss of ecosystem services and public attention to degradation subsides. In systems experiencing such societal acclimation to degradation, net incentives for stakeholder mitigation peak during early degradation phases and subside over time. Using harmful algae blooms in western Lake Erie as a case study, we illustrate how declines in public attention and societal reliance on lake recreation (i.e., finding recreation alternatives) reduce the incentives for stakeholders to reduce pollution runoff (i.e., mitigation efforts throughout the watershed). We then analyze how acclimation can affect a broad array of conservation challenges by developing a general socio-ecological model of societal response to degradation. We find that delays in initiating stakeholder-driven mitigation efforts can exponentially prolong restoration projects. Furthermore, when alleviating intense degradation relies upon voluntary commitments by many individuals, windows of opportunity for mitigation can be very limited because feedback loops of societal adaptation doom late restoration efforts to failure and lock human-environment systems into degraded states. These windows of opportunity can be particularly narrow when a) stakeholder mitigation requires supportive public opinion or b) even modestly valuable alternative services are available in degraded ecosystems. In such cases, maintaining undegraded human-environment regimes may hinge on quickly initiating stakeholder mitigation movements and allocating limited government conservation funds soon after degradation begins instead of spreading mitigation efforts out over decades. Such initiatives, regardless of whether acclimation is slow or rapid in a given system, also greatly accelerate the pace of environmental restoration.

Significance Statement

As societies acclimate to degraded environments, mitigation efforts that hinge on action by many stakeholders can erode. Developing a socio-ecological model of acclimation, we reveal how social and environmental processes intertwine to create alternative stable socio-ecological regimes, with either: 1) undegraded ecosystem states sustained by widespread mitigation adoption, or 2) degraded states where societies neither maintain nor continue relying on traditional, local ecosystem services. This dynamic places a premium on prompt mitigation efforts, which may face narrow opportunity windows to get started and avert degraded regimes in systems that rely on stakeholder-driven mitigation. Moreover, in any system requiring stakeholder action, societal acclimation will increase the importance of early action because decaying mitigation incentives exponentially lengthen restoration efforts.
Adaptive structural and functional evolution of the placenta protects fetal growth in high elevation deer mice
Authors: Wilsterman Kathryn , Moore Emily C. , Schweizer Rena M. , Cunningham Kirksey , Good Jeffrey M. , Cheviron Zachary A.
Published: September 2022

Environmental hypoxia challenges female reproductive physiology in placental mammals, increasing rates of gestational complications. Adaptation to high elevation has limited many of these effects in humans and other mammals, offering potential insight into the developmental processes that lead to and protect against hypoxia-related gestational complications. However, our understanding of these adaptations has been hampered by a lack of experimental work linking the functional, regulatory, and genetic underpinnings of gestational development in locally-adapted populations. Here, we dissect high-elevation adaptation in the reproductive physiology of deer mice, (Peromyscus maniculatus), a rodent species with an exceptionally broad elevational distribution that has emerged as a model for hypoxia adaptation. Using experimental acclimations, we show that lowland mice experience pronounced fetal growth restriction when challenged with gestational hypoxia, while highland mice maintain normal growth by expanding the compartment of the placenta that facilitates nutrient and gas exchange between dam and fetus. We then use compartmentspecific transcriptome analyses to show that adaptive structural remodeling of the placenta is coincident with widespread changes in gene expression within this same compartment. Genes associated with fetal growth in deer mice significantly overlap with genes involved in human placental development, pointing to conserved or convergent pathways underlying these processes. Finally, we overlay our results with genetic data from natural populations to identify can-didate genes and genomic features that contribute to these placental adaptations. Collectively, these experiments advance our understanding of adaptation to hypoxic environments by revealing physiological and genetic mechanisms that shape fetal growth trajectories under maternal hypoxia.

Significance Statement

Residence at high elevations is associated with higher risk pregnancies and low birth weight, yet the causal mechanisms remain poorly understood. Using a high elevation-adapted rodent model, we investigated the physiological traits that explain fetal growth trajectories in low oxygen environments, and how evolutionary adaptation has modified these traits. We showed that high- and low-elevation populations of deer mice differ in their susceptibility to fetal growth restriction during gestational hypoxia and that these population-level differences are associated with structural and transcriptomic changes in the placenta. We further link placental gene expression to genomic features under selection at high elevation. Our findings identify adaptations that are likely relevant to offsetting the effects of hypoxia on fetal and placental development across mammals.