bluuebunny/crossref_metadata_2025 huggingface dataset:
Photothermally Detected Stimulated Raman Microscopy towards Ultrasensitive Chemical Imaging
Authors: Zhu Yifan , Ge Xiaowei , Ni Hongli , Yin Jiaze , Lin Haonan , Wang Le , Tan Yuying , Prabhu Dessai Chinmayee V. , Cheng Ji-Xin
Published: March 2023

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The thereafter relaxation heats up the surrounding and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity.

One-Sentence Summary

We demonstrate a new spectroscopic imaging method that improves the signal intensity by >500-fold.
Zebrahub – Multimodal Zebrafish Developmental Atlas Reveals the State-Transition Dynamics of Late-Vertebrate Pluripotent Axial Progenitors
Authors: Lange Merlin , Granados Alejandro , VijayKumar Shruthi , Bragantini Jordão , Ancheta Sarah , Santhosh Sreejith , Borja Michael , Kobayashi Hirofumi , McGeever Erin , Solak Ahmet Can , Yang Bin , Zhao Xiang , Liu Yang , Detweiler Angela M. , Paul Sheryl , Mekonen Honey , Lao Tiger , Banks Rachel , Kim Yang-Joon , Jacobo Adrian , Balla Keir , Awayan Kyle , D’Souza Samuel , Haase Robert , Dizeux Alexandre , Pourquie Olivier , Gómez-Sjöberg Rafael , Huber Greg , Serra Mattia , Neff Norma , Pisco Angela Oliveira , Royer Loïc A.
Published: March 2023

Elucidating the developmental processes of organisms requires a comprehensive understanding of cellular lineages in the spatial, temporal, and molecular domains. In this study, we introduce Zebrahub, a dynamic atlas of zebrafish embryonic development that integrates single-cell sequencing time course data with lineage reconstructions facilitated by light-sheet microscopy. This atlas offers high-resolution and in-depth molecular insights into zebrafish development, achieved through the sequencing of individual embryos across ten developmental stages, complemented by trajectory reconstructions. Zebrahub also incorporates an interactive tool to navigate the complex cellular flows and lineages derived from light-sheet microscopy data, enablingin silicofate mapping experiments. To demonstrate the versatility of our multi-modal resource, we utilize Zebrahub to provide fresh insights into the pluripotency of Neuro-Mesodermal Progenitors (NMPs). Our publicly accessible web-based platform, Zebrahub, is a foundational resource for studying developmental processes at both transcriptional and spatiotemporal levels, providing researchers with an integrated approach to exploring and analyzing the complexities of cellular lineages during zebrafish embryogenesis.
The novel coronary artery disease risk factor ADAMTS-7 modulates atherosclerotic plaque formation by degradation of TIMP-1
Authors: Sharifi M. Amin , Wierer Michael , Dang Tan An , Milic Jelena , Moggio Aldo , Sachs Nadja , von Scheidt Moritz , Hinterdobler Julia , Müller Philipp , Werner Julia , Stiller Barbara , Aherrahrou Zouhair , Erdmann Jeanette , Zaliani Andrea , Graettinger Mira , Reinshagen Jeanette , Gul Sheraz , Gribbon Philip , Maegdefessel Lars , Bernhagen Jürgen , Sager Hendrik B. , Mann Matthias , Schunkert Heribert , Kessler Thorsten
Published: March 2023

Background

TheADAMTS7locus was genome-wide significantly associated with coronary artery disease (CAD). Lack of the extracellular matrix (ECM) protease ADAMTS-7 was shown to reduce atherosclerotic plaque formation.

Objective

To identify molecular mechanisms and downstream targets of ADAMTS-7 mediating risk of atherosclerosis.

Methods

Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence,in vitrodegradation assays, co-immunoprecipitation, and Förster resonance energy transfer (FRET)-based protein-protein interaction assays.ADAMTS7expression was measured in fibrous caps of human carotid artery plaques.

Results

In humans,ADAMTS7expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of tissue inhibitor of metalloproteases 1 (Timp-1). In co-immunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7in vitro.ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target matrix metalloprotease 9 (MMP-9) As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. In order to facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim to decrease TIMP-1 degradation, we designed a FRET-based assay targeting the ADAMTS-7 catalytic site.

Conclusion

ADAMTS-7,which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise genome-wide significantly associated with CAD. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for reduction of atherosclerosis-related events.
High Maternal Adiposity During Pregnancy Programs an Imbalance in the Lipidome and Predisposes to Diet-induced Hepatosteatosis in the Offspring
Authors: Scheidl Taylor B. , Wager Jessica L. , Baker Larissa G. , Brightwell Amy L. , Melan Katrina M. , Larion Sebastian , Sarr Ousseynou , Regnault Timothy RH. , Urbanski Stefan J , Thompson Jennifer A.
Published: March 2023

Background

Exposure to high maternal adiposityin uterois a significant risk factor for the later-life development of metabolic syndrome (MetS), including non-alcoholic fatty liver disease (NAFLD). We have previously shown that high pre-pregnancy adiposity programs adipose tissue dysfunction in the offspring, leading to spillover of fatty acids into the circulation, a key pathogenic event in obesity-associated MetS. Herein, we hypothesized that programming of adipose tissue dysfunction in offspring born to overweight dams increases the risk for developing NAFLD.

Results

Females heterozygous for leptin receptor deficiency (Hetdb) were used as a model of high pre-pregnancy adiposity. Wild-type (Wt) offspring born to Hetdbpregnancies gained significantly more body fat following high fat/fructose diet (HFFD) compared to Wt offspring born to Wt dams. HFFD increased circulating free fatty acids (FFA) in male offspring of control dams, while FFA levels were similar in HFFD-fed offspring from Wt dams compared to CD or HFFD-Wt offspring from Hetdbdams. Despite female-specific protection from diet-induced FFA spillover, both male and female offspring from Hetdb. dams were more susceptible to diet-induced hepatosteatosis. Lipidomic analysis revealed that CD-offspring of overweight dams had decreased hepatic PUFA levels compared to control offspring. Changes to saturated fatty acids (SFA) and thede novolipogenic (DNL) index were diet driven; however, there was a significant effect of the intrauterine environment on FA elongation and Δ9 desaturase activity.

Conclusion

High maternal adiposity during pregnancy programs a susceptibility to diet-induced hepatosteatosis.
Cryo-EM of α-tubulin isotype containing microtubules revealed a contracted structure of α4A/β2A microtubules
Authors: Diao Lei , Zheng Wei , Zhao Qiaoyu , Liu Mingyi , Fu Zhenglin , Zhang Xu , Bao Lan , Cong Yao
Published: March 2023

Microtubules are hollow α/β-tubulin heterodimeric polymers playing critical roles in cells. In vertebrates, both α- and β-tubulin have multiple isotypes encoded by different genes, which are intrinsic factors in regulating microtubule functions. However, structures of microtubules composed of different tubulin isotypes, especially α-tubulin isotypes, remain largely unknown. Here, we purified recombinant tubulin heterodimers composed of different mouse α-tubulin isotypes, including α1A, α1C and α4A, with β-tubulin isotype β2A. We further assembled and determined the cryo-electron microscopy (cryo-EM) structures of α1A/β2A, α1C/β2A, and α4A/β2A microtubules. Our structural analysis demonstrated that α4A/β2A microtubules exhibit a longitudinal contraction between tubulin interdimers compared with α1A/β2A and α1C/β2A microtubules. Collectively, our findings reveal that α-tubulin isotype composition could tune microtubule structures, and also provide evidence for the “tubulin code” hypothesis.
PERK-mediated induction of miR-5p and miR-3p arms of miR-616 regulates cell growth by targeting c-MYC
Authors: Arabkari Vahid , Sultana Afrin , Barua David , Webber Mark , Smith Terry , Gupta Ananya , Gupta Sanjeev
Published: March 2023

C/EBP homologous protein (CHOP), also known as DNA damage-inducible transcript 3 (DDIT3), is a member of CCAAT/enhancer-binding protein (C/EBP) family. The expression of CHOP is upregulated during unfolded protein response (UPR), and sustained CHOP activity plays an important role in UPR-induced apoptosis. MicroRNA-616 is localized in an intron of the CHOP gene. However, regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. We show that miR-5p/-3p arms of miR-616 are expressed with levels of 5p arm higher than 3p arm. During conditions of UPR, the expression of miR-5p and miR-3p arms of miR-616 and its host gene (CHOP) was concordantly increased primarily in a PERK-dependent manner. We show that ectopic expression of miR-616 significantly suppressed cell growth and colony formation, whereas knockout of miR-616 increased it. We identified that MYC proto-oncogene (c-MYC) gene is repressed during the UPR and targeted by miR-616. Further, we show that expression of miR-616 and CHOP is downregulated in human breast cancer, where expression of miR-616 was associated with poor overall survival (OS) in luminal A subtype and better OS HER2 subtype of breast cancer. In summary, our results suggest a dual function for the DDIT3 locus, where CHOP protein and miR-616 can co-operate to regulate cancer progression.
A bootstrap model comparison test for identifying genes with context-specific patterns of genetic regulation
Authors: Malakhov Mykhaylo M. , Dai Ben , Shen Xiaotong T. , Pan Wei
Published: March 2023

Understanding how genetic variation affects gene expression is essential for a complete picture of the functional pathways that give rise to complex traits. Although numerous studies have established that many genes are differentially expressed in distinct human tissues and cell types, no tools exist for identifying the genes whose expression is differentially regulated. Here we introduce DRAB (Differential Regulation Analysis by Bootstrapping), a gene-based method for testing whether patterns of genetic regulation are significantly different between tissues or other biological contexts. DRAB first leverages the elastic net to learn context-specific models of local genetic regulation and then applies a novel bootstrap-based model comparison test to check their equivalency. Unlike previous model comparison tests, our proposed approach can determine whether population-level models have equal predictive performance by accounting for the variability of feature selection and model training. We validated DRAB on mRNA expression data from a variety of human tissues in the Genotype-Tissue Expression (GTEx) Project. DRAB yielded biologically reasonable results and had sufficient power to detect genes with tissue-specific regulatory profiles while effectively controlling false positives. By providing a framework that facilitates the prioritization of differentially regulated genes, our study enables future discoveries on the genetic architecture of molecular phenotypes.
Medical student attitudes to patient involvement in healthcare decision-making and research
Authors: O'Neill Jennifer , Docherty Stewart Bronwyn , Ng Anna XN , Roy Yamini , Yousif Liena , McIntyre Kirsty R
Published: March 2023

Objective Patient involvement is used to describe the inclusion of patients as active participants in healthcare decision-making and research. This study aimed to investigate incoming Year 1 medical (MBChB) students attitudes and opinions regarding patient involvement in this context. Methods We established a staff-student partnership to formulate the design of an online research survey, which included Likert scale questions and three short vignette scenarios designed to probe student attitudes towards patient involvement linked to existing legal precedent. Incoming Year 1 medical students (n = 333) were invited to participate in the survey before formal teaching commenced. Results Survey data (49 participants) indicate that students were broadly familiar with, and supportive of, patient involvement in medical treatment. There was least support for patient involvement in conducting (22.4%), contributing to (34.7%) or communicating research (30.6%), whereas there was unanimous support for patients choosing treatment from a selection of options (100%). Conclusion Incoming members of the medical profession demonstrate awareness of the need to actively involve patients in healthcare decision-making but are unfamiliar with the utility and value of such involvement in research. Further empirical studies are required to examine attitudes to patient involvement in healthcare.
Hydrogen sulfide small intestinal bacterial overgrowth case registry
Authors: Goldenberg Joshua Z , Nevitt Britta , Wentz Anna E , Bradley Ryan , Siebecker Allison
Published: March 2023

Background

There is growing interest in hydrogen sulfide small intestinal bacterial overgrowth (H2S SIBO). However, basic questions including how clinicians are making the diagnosis, what symptoms are present, and what clinicians are using for treatment, remain unanswered.

Aims

To address this, we created an online, survey-based, clinical registry of H2S SIBO cases.

Methods

Participants in this study were clinicians diagnosing and treating patients with H2S SIBO and input data on patient presentation, diagnosis, treatment, and treatment response. We describe the population and summarize our results using descriptive statistics. We use Pearson’s chi-squared test and modified Poisson regression in exploratory analyses.

Results

131 total cases were submitted by 99 providers across a spectrum of health disciplines. The mean patient age was 45.6 (71.4% female). The most common symptoms were bloating (77.0%), constipation (50.8%) and abdominal pain (50.8%). Diagnosis was made based on flatline hydrogen in the 3rd hour of a lactulose breath test (42.5%), symptom presentation (empiric) (31.9%), or H2S levels (25.7%). The most common treatments used were a low sulfur diet (46.6%), oregano (44.0%), and bismuth (39.7%). Fifty-eight percent of cases were responders. Of the most common interventions used, only a low sulfur diet (73% responder; p=0.01) and bismuth (76% responder; p=0.01) were significantly associated with treatment response. Interestingly, response rates differed based on how H2S SIBO was diagnosed, with empiric underperforming flatline diagnoses (relative risk 0.60; p=0.04).

Conclusions

This case registry represents the largest collection of H2S SIBO cases to-date, providing important early descriptive information on this emerging diagnosis.
Gentrification and Food Environments: A Rapid Evidence Assessment
Authors: Gie Simone , Borthwick Fiona
Published: March 2023

Gentrification is a complex and controversial process, where the influx of new, wealthier residents to previously run-down neighbourhoods brings change such as economic development, infrastructure investments and lower crime rates, but can be to the detriment of the original lower-income residents, who are either displaced, or stay but cannot take advantage of the new opportunities. Understanding how neighbourhood change affects food environments can shed light on the possible causal pathways between gentrification and urban health inequalities. This rapid evidence assessment reviewed evidence on the impact of gentrification on the healthfulness of food environments globally. Ten studies were identified through a systematic keyword search and assessed. We found limited evidence of an effect, with a small, albeit consistent, body of evidence mostly comprised of low- to medium-quality observational studies, all from high-income countries. Most studies examined effects on availability or affordability of food, finding an association between gentrification and increased availability of unhealthy foods, or reduced affordability for original low-income residents.
The impact of public-initiated COVID-19 risk communication on individual NPI practices
Authors: Tefera Yifokire , Kumie Abera , Hailemariam Damen , Wakuma Samson , Abegaz Teferi , Yirsaw Shibabaw
Published: March 2023

Background

Non-Pharmaceutical Interventions (NPI) are the most widely recognized public health measures practiced globally to prevent the spread of Covid-19 transmission. The effectiveness of NPIs is dependent on the type, a combination of applied interventions, and the level of compliance of the NPIs. The expected outcome of behavioral practices varies relative to the behavioral intervention duration.

Objectives

This study aimed to assess the trend of community compliance to NPIs and with its level of variation with the place of residence and sociodemographic characteristics of people.

Methods

A weekly non-participatory field survey on an individual’s NPI practice was observed from October 2020 to July 2021, for a total of 39 weeks. The survey covered all the regions; 14 regional and capital cities. Data collection for the 3 NPI behaviors (mask use, hand hygiene, and physical distance was and managed weekly at eight public service locations using the Open Data Kit (ODK) tool.

Results

More than 180,000 individuals were observed for their NPI practice; on average 5,000 observations in a week. About 43% of the observation was made from Addis Ababa, 56% were male participants and the middle age group (18-50 years) accounts for 75%. The overall level of NPI compliance had a peak around the 26th– 30thweeks then decline the rest of the weeks. Respiratory hygiene had the highest compliance whereas hand hygiene had the least 41% and 4%, respectively. There was a significant difference between capital city and regional city residents by their level of NPI compliance. Females comply more than males, and individuals had increased NPI compliance while at the bank service and workplaces compared to while in the transport services at p<0.001.

Conclusion

The overall community compliance with NPI practice showed a declining trend in Ethiopia but increased compliance was also observed following the implementation of government-initiated public measures. Therefore, public-initiated risk communication and public advocacy programs for the prevention of Covid-19 should be strengthened.
Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins
Authors: Cannon Kevin S. , Sarsam Reta D. , Tedamrongwanish Tanita , Zhang Kevin , Baker Richard W.
Published: March 2023

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.
The nucleolar aberrancies that drive ribosome impairment induced by RNA binding proteins are hallmarks of aging
Authors: Aguilar-Garrido Pedro , Velasco-Estévez María , Navarro-Aguadero Miguel Ángel , Hernandez-Sanchez María , Malaney Prerna , Zhang Xiaroui , Aitken Marisa J. L. , Otero-Sobrino Alvaro , Ibañez-Navarro Marta , Ortiz-Ruiz Alejandra , Megias Diego , Pérez-Martínez Manuel , Mata Gadea , Gomez Jesús , Dominguez Orlando , Graña-Castro Osvaldo , Caleiras Eduardo , de Andres Paloma Jimena , Rodriguez Sandra , Torres Raúl , Sirozh Oleksandra , Lafarga Vanesa , Martinez-Lopez Joaquín , Post Sean M. , Gallardo Miguel
Published: March 2023

The nucleolus is a dynamic structure where ribosome subunits are produced. Indeed, nucleoli respond to any change in cellular homeostasis by altering the rate of ribosome biogenesis, thus working as a stress sensor. Therefore, an imbalance in ribosome biogenesis promotes changes in morphology and function and can evoke a nucleolar stress response. Changes in the structure and composition of nucleoli impair ribosome biogenesis and have been described as nucleolar stress, a mechanism related to aging and cancer.

Here, we show the role of the RNA binding protein Hnrnpk in nucleolar dynamics and ribosome function. Hnrnpk is a ribonucleoprotein in charge of escorting nascent transcripts to its processing and nuclear export to ribosomes. When Hnrnpk is overexpressed, the nucleolus is altered and shows stress-like phenotype, with accumulation and delocalization of components such as Ncl, driving ribosome biogenesis impairment and halting protein translation.

Nucleolin haploinsufficiency is correlated with enlarged nucleoli, increased ribosome components and translation and induces a reduction in lifespan. Thus, gain of Ncl generated by Hnrnpk overexpression can cause ribosome biogenesis defects associated with ribosome impairment leading to ribosomopathies and bone marrow failure syndrome.

Aging and bone marrow failure share common biological hallmarks. Indeed, Hnrnpk overexpression and nucleolar stress trigger cell cycle arrest and senescence of the cells, a feature of both processes.

Together, these findings support the idea that nucleolar abnormalities contribute to ribosome impairment, thus triggering the onset of hematopoiesis and the aging process. Here, we decipher a novel master regulator of this mechanism: Hnrnpk.
Tmem178 negatively regulates IL-1β production through inhibition of the NLRP3 inflammasome
Authors: Khanna Kunjan , Yan Hui , Mehra Muneshwar , Rohatgi Nidhi , Mbalaviele Gabriel , Faccio Roberta
Published: March 2023

Objective

Inflammasomes modulate the release of bioactive IL-1β. Excessive IL-1β levels are detected in patients with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS) with mutated and unmutated inflammasome components, raising questions on the mechanisms of IL-1β regulation in these disorders.

Methods

To investigate how the NLRP3 inflammasome is modulated in sJIA, we focused on Tmem178, a negative regulator of calcium levels in macrophages, and measured IL-1β and caspase-1 activation in wild-type (WT) andTmem178-/-macrophages following calcium chelators, silencing of Stim1, a component of store-operated calcium entry (SOCE), or by expressing a Tmem178 mutant lacking Stim1 binding site. Mitochondrial function in both genotypes was assessed by measuring oxidative respiration, mitochondrial reactive oxygen species (mtROS), and mitochondrial damage. CSS development was analyzed inPerforin-/-/Tmem178-/-mice infected with LCMV in which inflammasome or IL-1 signaling was pharmacologically inhibited. HumanTMEM178andIL-1Btranscripts were analyzed in a dataset of peripheral blood monocytes from healthy controls and active sJIA patients.

Results

TMEM178levels are reduced in monocytes from sJIA patients while IL-1B show increased levels. Accordingly,Tmem178-/-macrophages produce elevated IL-1β compared to WT cells. The elevated intracellular calcium levels following SOCE activation inTmem178-/-macrophages induce mitochondrial damage, release mtROS, and ultimately, promote NLRP3 inflammasome activation.In vivo, inhibition of inflammasome or IL-1 neutralization prolongsTmem178-/-mouse survival to LCMV-induced CSS.

Conclusion

Downregulation of Tmem178 levels may represent a new biomarker to identify sJIA/CSS patients that could benefit from receiving drugs targeting inflammasome signaling.
Identification of mobile element insertion from whole genome sequencing data using deep neural network model
Authors: Bu Fengxiao , Xu Xiaofei , Huang Yu , Wang Xuegang , Cheng Jing , Yuan Huijun
Published: March 2023

Mobile element insertions (MEIs) are a major contributor to genome evolution and play an essential role in the regulation of gene expression, as well as being implicated in various human diseases. This study introduces DeepMEI, a tool based on a convolutional neural network model that transforms the MEI identification process into an image recognition problem and automatically learns complex and abstract representations of MEI features in whole genome sequencing data. DeepMEI outperformed existing tools in the benchmark dataset from the Genome in a Bottle consortium, with a precision of 0.90 and recall of 0.70. Moreover, factors such as sequencing depth, ME integrity, and genome mappability can affect MEI identification accuracy. Using DeepMEI, we reanalyzed 3,202 high-coverage whole-genome sequencing samples from the 1000 Genome Project (1kGP) phase 4 release, discovering 1.71-fold more non-reference MEIs, totaling 6,218,088, with 92.2% of the increase coming from rare MEIs (allele frequency <1%). This enhances our understanding of MEIs role in human disease and evolution. The DeepMEI tool and the updated 1kGP MEI dataset can be accessed at https://github.com/xuxif/DeepMEI.
Deciphering dermal fibroblast behavior in 3D bioprinted dermis contructs
Authors: Chastagnier Laura , el-Kholti Naima , Essayan Lucie , Thomann Céline , Courtial Edwin-Joffrey , Marquette Christophe , Petiot Emma
Published: March 2023

In recent years, numerous strategies have emerged to answer the growing demand for graftable tissues. Tissue engineering andin-vitroproduction are one of them. Among all the engineered tissues, skin is one of the most advanced. Nevertheless, biofabrication of graftable and fully functional skin substitutes is still far from being reached. Skin reconstruction, particularly dermis, necessitates cultivation and maturation for several weeks (> 3 weeks) to recover the tissue’s composition and functions, which prevent its transfer to clinical applications. Thus, several strategies, including 3D bioprinting, have been explored to accelerate these productions. In the present study, based on the successful application of 3D bioprinting achieved by our group for skin reconstruction in 21 days, we propose to detail the biological behaviors and maturation phases occurring in the bioprinted skin construct thanks to a descriptive approach transferred from the bioprocess field. The aim is to comprehensively characterize dermis construct maturation phases (cell proliferation and ECM secretion) to master later the interdependent and consecutive mechanisms involved inin-vitroproduction. Thus, standardized quantitative techniques were deployed to describe 3D bioprinted dermis proliferation and maturation phases. Then, in a second step, various parameters potentially impacting the dermis reconstruction phases were evaluated to challenge our methodology and reveal the biological behavior described (fibroblast proliferation and migration, cell death, ECM remodeling with MMP secretion). The parameters studied concern the bioprinting practice including various printed geometries, bioink formulations and cellular physiology in relation with their nutritional supplementation with selected medium additives.
A Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of trypanosomes
Authors: Gerber Markus , Suppanz Ida , Oeljeklaus Silke , Niemann Moritz , Käser Sandro , Warscheid Bettina , Schneider André , Dewar Caroline E.
Published: March 2023

The AAA-ATPase Msp1 extracts mislocalized outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pull down experiments we show that trypanosomal Msp1 localizes to both glycosomes and the mitochondrial outer membrane, where it forms a stable complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of a-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double and triple RNAi cell lines combined with proteomic analyses we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates suggesting they act in concert with Msp1. pATOM36 is a functional analogue of the yeast MIM complex and possibly of human MTCH2 suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.
SIN3 acts in distinct complexes to regulate the germline transcriptional program inC. elegans
Authors: Caron M. , Robert V. , Gely L. , Adrait A. , Pakulska V. , Couté Y. , Chevalier M. , Riedel C.G. , Bedet C. , Palladino F.
Published: March 2023

The SIN3 transcriptional coregulator influences gene expression through multiple interactions that include histone deacetylases (HDACs). Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability (ID)/autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here we show that loss of SIN-3, the single SIN3 homologue inCaenorhabditis elegans, results in maternal effect sterility associated with deregulation of the germline transcriptome, including desilencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific HDACs, and show that they differentially contribute to fertility. Single cell smFISH reveals that insin-3mutants, the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN3. Together, this work provides a powerful framework for thein vivostudy of SIN3 and associated proteins.
Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells
Authors: Morgun Eva , Zhu Jennifer , Almunif Sultan , Bobbala Sharan , Aguilar Melissa S. , Wang Junzhong , Conner Kathleen , Cui Yongyong , Cao Liang , Seshadri Chetan , Scott Evan A. , Wang Chyung-Ru
Published: March 2023

Mycobacterium tuberculosis(Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA forin vivodelivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation andin vivoactivation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.
Landing force reveals new form of motion-induced sound camouflage in a wild predator
Authors: Schalcher Kim , Milliet Estelle , Séchaud Robin , Bühler Roman , Almasi Bettina , Potier Simon , Becciu Paolo , Roulin Alexandre , Shepard Emily L. C.
Published: March 2023

Predator-prey arms races have led to the evolution of finely-tuned disguise strategies. While the theoretical benefits of predator camouflage are well established, no study has yet been able to quantify its consequences for hunting success in natural conditions. We used high-resolution movement data to quantify how barn owls (Tyto alba) conceal their approach when using a sit-and-wait strategy. We hypothesized that hunting barn owls would modulate their landing force, potentially reducing noise levels in the vicinity of prey. Analysing 87,957 landings by 163 individuals equipped with GPS tags and accelerometers, we show that barn owls reduce their landing force as they approach their prey, and that landing force predicts the success of the following hunting attempt. Landing force also varied with the substrate, being lowest on man-made poles in field boundaries. The physical environment therefore affects the capacity for sound camouflage, providing an unexpected link between predator-prey interactions and land-use. Finally, hunting strike forces in barn owls were the highest recorded in any bird, relative to body mass, highlighting the range of selective pressures that act on landings and the capacity of these predators to modulate their landing force. Overall, our results provide the first measurements of landing force in a wild setting, revealing a new form of motion-induced sound camouflage and its link to hunting success.
The FSGS disease gene product and nuclear pore protein NUP205 regulates nuclear localization and activity of the transcriptional regulators YAP and TAZ in podocytes
Authors: Ester Lioba , Cabrita Inês , Ventzke Michel , Christodoulou Marita , Fabretti Francesca , Benzing Thomas , Habbig Sandra , Schermer Bernhard
Published: March 2023

Background

Mutations in genes encoding nuclear pore proteins (NUPs) cause steroid-resistant nephrotic syndrome (SRNS) and focal and segmental glomerulosclerosis (FSGS). The mechanisms of how NUP deficiency may cause podocyte dysfunction and failure of the kidney filtration barrier are elusive. The tightly controlled activity of the transcriptional effectors of the evolutionarily conserved Hippo pathway YAP and TAZ is essential for podocyte homeostasis. Here we analyze the role of NUPs in controlling YAP/TAZ nuclear import and activity in podocytes.

Methods

We used quantitative label-free mass spectrometry to characterize the YAP/TAZ interactomes in podocytes, particularly identifying NUP interactions. Moreover, we specifically studied NUP205 in controlling YAP/TAZ nuclear import and YAP/TAZ-dependent target gene expression.

Results

Here we identify the disease-causing nuclear pore proteins NUP107, NUP133, NUP205, and XPO5 as components of YAP and TAZ protein complexes in podocytes. We demonstrate that NUP205 is essential for YAP/TAZ nuclear import. The nuclear interaction of YAP/TAZ with TEAD1 and their transcriptional activity were dependent on NUP205 expression. Furthermore, we identify a feedback regulatory mechanism that controls YAP activity depending on TAZ-mediated NUP205 expression.

Conclusion

This study links the disease protein NUP205 with the activity of the transcriptional regulators and Hippo effectors YAP and TAZ and suggests a pathogenic role of YAP/TAZ-deregulation in podocytes in patients withNUP205mutations. Moreover, this study suggests an important role of YAP/TAZ signaling in human FSGS.

SIGNIFICANCE STATEMENT

Understanding the interference of signaling pathways in genetic diseases is essential to finally develop tailored treatment strategies. The increasing number of pathogenic variants causing focal-segmental glomerulosclerosis (FSGS) comprises genes encoding for proteins of the nuclear-cytoplamic shuttling machinery. We here found several of these proteins in an unbiased interactome analysis of the Hippo signaling effector proteins YAP and TAZ in podocytes, pointing to a connection between nucleoporins and the Hippo pathway. Further analyses confirmed that NUP205 is essential for correct YAP/TAZ shuttling; depletion of NUP205 resulted in cytoplasmic retention and inactivation of YAP/TAZ. We suggest the nuclear pore and hippo signaling as a pathogenic module in FSGS.
The pleiotropic functions of Pri smORF peptides synchronise leg development regulators
Authors: Markus Damien , Pelletier Aurore , Boube Muriel , Port Filip , Boutros Michael , Payre François , Obermayer Benedikt , Zanet Jennifer
Published: March 2023

The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed inDrosophilaimaginal leg discs. Thanks to a functional screen in legs, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis andpriwas known to control leg development through misunderstood mechanisms, we investigated the role of Ubr3 in mediatingprifunction in legs. We found thatpriplay several roles during leg development both in patterning and in cell survival. At larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Earlier, during larval stage,priactivates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signalling pathways. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development.

Summary statement

Pri smORF peptides activate multiple actors regulating signalling pathways, transcription and apoptosis by distinct mechanisms to insure tarsal patterning and epithelial morphogenesis during leg development.
Tld1 is a novel regulator of triglyceride lipolysis that demarcates a lipid droplet subpopulation
Authors: Speer Natalie Ortiz , Braun R. Jay , Reynolds Emma Grace , Brudnicka Alicja , Swanson Jessica M.J. , Henne W. Mike
Published: March 2023

Cells store lipids in the form of triglyceride (TG) and sterol-ester (SE) in lipid droplets (LDs). Distinct pools of LDs exist, but a pervasive question is how proteins localize to and convey functions to LD subsets. Here, we show the yeast protein YDR275W/Tld1 (for TG-associated LD protein 1) localizes to a subset of TG-containing LDs, and reveal it negatively regulates lipolysis. Mechanistically, Tld1 LD targeting requires TG, and is mediated by two distinct hydrophobic regions (HRs). Molecular dynamics simulations reveal Tld1’s HRs interact with TG on LDs and adopt specific conformations on TG-rich LDs versus SE-rich LDs in yeast and human cells. Tld1-deficient yeast display no defect in LD biogenesis, but exhibit elevated TG lipolysis dependent on lipase Tgl3. Remarkably, over-expression of Tld1, but not LD protein Pln1/Pet10, promotes TG accumulation without altering SE pools. Finally, we find Tld1-deficient cells display altered LD mobilization during extended yeast starvation. We propose Tld1 senses TG-rich LDs and regulates lipolysis on LD subpopulations.
Bacterial cGAS senses a viral RNA to initiate immunity
Authors: Banh Dalton V. , Roberts Cameron G. , Amador Adrian Morales , Brady Sean F. , Marraffini Luciano A.
Published: March 2023

CBASS immunity protects prokaryotes from viral (phage) attack through the production of cyclic dinucleotides which activate effector proteins that trigger the death of the infected host. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a highly structured 400-nt RNA, termedCBASS-activatingbacteriophage RNA (cabRNA), that binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP. Phages that escape CBASS immunity harbor mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. Since the mammalian cyclase OAS1 also binds viral dsRNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defense pathways.
Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease
Authors: Zaver Shivam A. , Sarkar Mrinal K. , Egolf Shaun , Zou Jonathan , Tiwaa Afua , Capell Brian C. , Gudjonsson Johann E. , Simpson Cory L.
Published: March 2023

Mutation of theATP2A2gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than two decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. SinceAtp2a2knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomic analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAP kinase signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings with lesions showing keratin deficiency, cadherin mis-localization, and ERK hyper-phosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multi-omic analysis with human organotypic epidermis as a pre-clinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.
Intrinsically disordered protein’s coil-to-globule transition and adsorption onto a hydrophobic surface under different conditions
Authors: Faulí Bernat Durà , Bianco Valentino , Franzese Giancarlo
Published: March 2023

Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) can modulate cellular responses to environmental conditions by undergoing coil-to-globule transitions and phase separation. However, the molecular mechanisms of these phenomena still need to be fully understood. Here, we use Monte Carlo calculations of a model incorporating water’s effects on the system’s free energy to investigate how an IDP responds to a hydrophobic surface under different conditions. We show that a slit pore confinement without top-down symmetry enhances the unfolding and adsorption of the IDP in both random coil and globular states. Moreover, we demonstrate that the hydration water modulates this behavior depending on the thermodynamic parameters. Our findings provide insights into how IDPs and IDRs can sense and adjust to external stimuli such as nanointerfaces or stresses.
Machine learning reveals relationships between song, climate, and migration in coastalZonotrichia leucophrys
Authors: Yang Jiaying , Carstens Bryan C. , Provost Kaiya L.
Published: March 2023

Vocalization behavior in birds, especially songs, strongly affects reproduction, but it is also highly impacted by geographic distance, climate, and time. For this reason, phenotypic differences in vocalizations between different bird populations are often interpreted as evidence of lineage divergence. Previous work has demonstrated that there is extensive variation in the songs of White-crowned Sparrow (Zonotrichia leucophrys) throughout the species range, including between neighboring (and genetically distinct) subspeciesZ. l. nuttalliandZ. l. pugetensis. However, it is unknown whether the divergence in their songs correlates to environmental or geographical factors. Previous work has been hindered by time-consuming traditional methods to study bird songs that rely on the manual annotation of song spectrograms into individual syllables. Here we explore the performance of automated machine learning methods of song annotation, which can process large datasets more efficiently, paying attention to the question of subspecies differences. We utilize a recently published artificial neural network to automatically annotate hundreds of White-crowned Sparrow vocalizations across two subspecies. By analyzing differences in syllable usage and composition, we find thatZ. l. nuttalliandZ. l. pugetensishave significantly different songs. Our results are consistent with the interpretation that these differences are caused by the changes in syllables in the White-crowned Sparrow repertoire. However, the large sample size enabled by the AI approach allows us to demonstrate that divergence in song is correlated with environmental difference and migratory status, but not with geographical distance. Our findings support the hypothesis that the evolution of vocalization behavior is affected by environment, in addition to population structure.

LAY SUMMARY

Birdsong is an important behavior because it is important in bird communication and reproduction.

White-crowned Sparrows in western North America are known to use different songs along their range, but it is unknown if those songs vary due to the environment.

We used machine learning to analyze these songs and found that populations of White-crowned Sparrows can be differentiated based on their songs.

Environmental factors during the breeding season exert a greater influence on song evolution in migratory subspecies.