bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted engagement of β-catenin-Ikaros complexes in refractory B-cell malignancies
Authors: Cosgun Kadriye Nehir , Jumaa Huda , Robinson Mark E. , Kistner Klaus M. , Xu Liang , Xiao Gang , Chan Lai N. , Lee Jaewoong , Kume Kohei , Leveille Etienne , Fonseca-Arce David , Khanduja Dhruv , Ng Han Leng , Feldhahn Niklas , Song Joo , Chan Wing-Chung , Chen Jianjun , Taketo M. Mark , Kothari Shalin , Davids Matthew S. , Schjerven Hilde , Jellusova Julia , Müschen Markus
Published: March 2023

In most cell types, nuclear β-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptionalactivationof MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of β-catenin but critically depended on GSK3β for effective β-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, β-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptionalrepressionof MYC.

To leverage this previously unrecognized vulnerability of B-cell-specific repressive β-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3β small molecule inhibitors to subvert β-catenin degradation. Clinically approved GSK3β-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of β-catenin, repression of MYC and acute cell death. Preclinicalin vivotreatment experiments in patient-derived xenografts validated small molecule GSK3β-inhibitors for targeted engagement of lymphoid-specific β-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies.

HIGHLIGHTS

Unlike other cell lineages, B-cells express nuclear β-catenin protein at low baseline levels and depend on GSK3β for its degradation.

In B-cells, β-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes.

CRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoidMYCsuperenhancer region reversed β-catenin-dependent Myc repression and induction of cell death.

The discovery of GSK3β-dependent degradation of β-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3β-inhibitors for the treatment of refractory B-cell malignancies.

GRAPHICAL ABSTRACT

Abundant nuclear β-catenin

β-catenin pairs with TCF7 factors for transcriptional activation of MYC

B-cells rely on efficient degradation of β-catenin by GSK3β

B-cell-specific expression of Ikaros factors

Unique vulnerability in B-cell tumors:

GSK3β-inhibitors induce nuclear accumulation of β-catenin.

β-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC
Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome
Authors: Venkatraman Kailash , Lee Christopher T. , Garcia Guadalupe C. , Mahapatra Arijit , Milshteyn Daniel , Perkins Guy , Kim Keun-Young , Pasolli H. Amalia , Phan Sebastien , Lippincott-Schwartz Jennifer , Ellisman Mark H. , Rangamani Padmini , Budin Itay
Published: March 2023

Cristae are high curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous mechanisms for lipids have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the IMM against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. The model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that CL is essential in low oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of CL is dependent on the surrounding lipid and protein components of the IMM.

Synopsis

critical lipidic breakpoint for yeast mitochondria phenocopies the loss of cristae-shaping proteins in the IMM.

saturation controls membrane mechanical properties and modulates ATP synthase oligomerization.

mitochondrial-specific lipid cardiolipin can functionally compensate for increased phospholipid saturation and is required for cristae formation in low oxygen environments.

mathematical model for cristae membrane tubules predicts a snapthrough instability mediated by both protein and lipid-encoded curvatures.

Synopsis Figure
Modelling collagen fibril self-assembly from extracellular medium in embryonic tendon
Authors: Revell Christopher K. , Herrera Jeremy A. , Lawless Craig , Lu Yinhui , Kadler Karl E. , Chang Joan , Jensen Oliver E.
Published: March 2023

Collagen is a key structural component of multicellular organisms and is arranged in a highly organised manner. In structural tissues such as tendons, collagen forms bundles of parallel fibres between cells, which appear within a 24 hour window between E13.5 and E14.5 during mouse embryonic development. Current models assume that the organised structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time- and length-scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the intercellular spaces prior to the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in intercellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.
Stroke-related gene expression profiles across species: A meta-analysis
Authors: Rust Ruslan
Published: March 2023

Stroke patients are often left with permanent disabilities with no regenerative treatment options. Unbiased RNA sequencing studies decoding the transcriptional signature of stroked tissue hold promise to identify new potential targets and pathways directed to improve treatment for stroke patients. Here, gene expression profiles of stroked tissue across different time points, species, and stroke models were compared using NCBI GEO database. In total, 32 datasets from mice, rats, humans, and primates were included, exploring gene expression differences in healthy and stroked brain tissue. Distinct changes in gene expression and pathway enrichment revealed the heterogenicity of the stroke pathology in stroke-related pathways e.g., inflammatory responses, vascular repair, remodelling and cell proliferation and adhesion but also in diverse general, stroke-unrelated pathways that have to be carefully considered when evaluating new promising therapeutic targets.
Molecular bases for strong phenotypic effects of single-site synonymous substitutions in theE.coli ccdBtoxin gene
Authors: Bajaj Priyanka , Bhasin Munmun , Varadarajan Raghavan
Published: March 2023

Single-site synonymous mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~200 single-site synonymous mutations in an operonic context by mutating each position ofccdB, the 101-residue long cytotoxin of theccdABToxin-Antitoxin (TA) operon to all possible degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistantE. colistrains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained forccdBsynonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed. Synonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, transcript secondary structure, and evolutionary conservation in determining phenotypic effects ofccdBsynonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single-site synonymous mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate but might also alter the folding kinetics of the proteinin vivo. This information is useful in optimizing heterologous gene expression inE. coliand understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations.

SIGNIFICANCE STATEMENT

Synonymous mutations which do not change amino-acid identity, typically have only minor or no effects on gene function. Using sensitive genetic screens in the context of theccdABbacterial toxin-antitoxin operon, we demonstrate that many single-site synonymous mutations of theccdBtoxin gene display significant phenotypic effects in an operonic context. The largest effects were seen for synonymous mutations in the N-terminal region involved in translation initiation. Synonymous mutations that affected either folding or translation termination were also identified. Lack of translational pausing due to synonymous mutations in hydrophobic residue stretches, was found to decrease the amount of properly folded CcdB protein. In summary the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function.
Left ventricular global longitudinal strain as a parameter of mild myocardial dysfunction in athletes after COVID-19
Authors: Schellenberg J , Ahathaller M , Matits L , Kirsten J , Kersten J , Steinacker JM
Published: March 2023

Background

Whether impaired left ventricular (LV) function contributes to persistent cardiopulmonary symptoms or decreased exercise capacity after COVID-19 remains unclear. The aim of this prospective study was to determine differences in LV global longitudinal strain (GLS) between athletes who did not have a history of LV dysfunction but had a positive COVID-19 test (PCAt) and healthy control (CON) athletes and relate them to symptoms during COVID-19.

Methods

We performed 151 transthoracic echocardiographies in our high-performance laboratory. GLS was determined in four-, two-, and three-chamber views and assessed offline by a blinded investigator in 88 PCAt (35% women) at a median of two months after COVID-19 who trained at least three times per week with more than 20 MET per week and 52 CONs from the German national squad (38% women).

Results

GLS was significantly lower (GLS -18.53±1.94% vs. -19.94±1.42%, p<0.001) and diastolic function significantly reduced (E/A 1.54±0.52 vs. 1.66±0.43, p=0.020; E’l 0.15±0.04 vs. 0.17±0.04, p=0.009; E/E’l 5.74±1.74 vs. 5.22±1.36, p=0.024) in PCAt. There was no association between GLS and acute symptoms like resting dyspnea, exertional dyspnea during or after COVID-19, palpitations, chest pain or increased resting heart rate. However, there was a trend toward lower GLS in PCAt with subjectively perceived performance limitation (p=0.054).

Conclusions

In a cohort of athletes at a median two months after COVID-19, significantly lower GLS and diastolic function were observed, suggesting mild myocardial dysfunction. GLS could be used as a screening element during return-to-sport examinations.
Association of radiological severity with inflammatory biomarkers for prognostic prediction in patients with COVID-19
Authors: Zouine Y. , Benzalim M. , Alj S.
Published: March 2023

Background

Covid – 19 caused by SARS-CoV2 has become a pandemic. It has a rapid disease progression and causes severe and fatal complications. Associating radiological severity with effective biomarkers like CRP, leucocytes, lymphocytes, D – Dimer, would be helpful in screening, categorizing patient, preventing serious complications.

Aims and Objectives

The aim of the study was to investigate association between levels of inflammatory biomarkers and correlate it with HRCT chest finding to identify patients at risk of fatal complications.

Materials and Methods

It was a retrospective monocentric observational study undertaken at Ibn Tofail hospital COVID-19 dedicated center. 177 Patients>18 year of age who were admitted from september 1, 2020 up to november 30,2020 with laboratory confirmed diagnosis of Covid – 19 were included in the study. Data was collected on demography, disease severity, laboratory measurements, radiology imaging retrospectively from records of patients. The disease severity was classified into light, mild to severe and critic based on CT Severity scoring. HRCT Chest and inflammatory biomarkers were sent in every patient at the time of admission and the outcome was recorded.

Results

There were 116 male patients, 61 female patients in our study. Average age of patients having severe lung involvement is 61.9years, whereas Average age of patients having non-severe lung involvement is 56.8 years and showed significant association with severity of lung involvement (p value : 0.017). Severity of lung involvement according to HRCT chest findings was greater in patients with both raised values of CRP <0.001), D – Dimer (P value 0.032) and low values of lymphocytes (P value : 0.001). Capillary oxygen SATURATION was also found to be significantly associated with radiological severity among covid-19 patients. Compared with CRP, leukocytes, lymphocytes, and D-Dimeres levels, the CT severity score had higher sensitivity, specificity, and overall accuracy in predicting severe, critical cases, and short-term mortality.

Conclusion

the severity of Covid – 19 disease is correlated with radiological severity andinflammatory markers thereby it will help in immediate categorization of patients into different risk groups following diagnosis, to ensure optimal resource allocation.
Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in theChlamydia trachomatisdevelopmental cycle
Authors: Shen Li , Gao Leiqiong , Swoboda Abigail R. , Ouellette Scot P.
Published: March 2023

Chlamydia trachomatisis an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes toChlamydiadevelopmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomaltopAtranscription inC. trachomatiswithout detected toxicity of dCas12. Repression oftopAimpaired the growth ofC. trachomatismostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes ofC. trachomatiswas downregulated while early genes maintained their expression. Importantly, the growth defect associated withtopAknockdown was rescued by overexpressingtopAat an appropriate degree and time, directly linking the growth patterns to the levels oftopAexpression. Interestingly,topAknockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency.C. trachomatiswithtopAknocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes ofC. trachomatis.

Importance

We used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential genetopAwith a CRISPRi approach, using dCas12, inC. trachomatisindicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanism(s) by which well-balanced topoisomerase activities enableC. trachomatisto adapt to unfavorable growth conditions imposed by antibiotics.
Deep physico-chemical characterization of the serum antibody response using a dual-titration microspot assay
Authors: Kovács Ágnes , Hérincs Zoltán , Papp Krisztián , Kaczmarek Jakub Zbigniew , Larsen Daniel Nyberg , Stage Pernille , Bereczki László , Ujhelyi Eszter , Pfeil Tamás , Prechl József
Published: March 2023

Antigen specific humoral immunity can be characterized by the analysis of serum antibodies. While serological assays for the measurement of specific antibody levels are available, these are not quantitative in the biochemical sense. Yet, understanding humoral immune responses quantitatively on the systemic level would need a universal, complete, quantitative, comparable measurement method of antigen specific serum antibodies of selected immunoglobulin classes. Here we describe a fluorescent, dual-titration immunoassay, which provides the physico-chemical parameters that are both necessary and sufficient to quantitatively characterize the humoral immune response. We define the theory of the approach that is based on physical chemistry. For validation of theory, we used recombinant receptor binding domain of SARS-CoV-2 as antigen on microspot arrays and varied the concentration of both the antigen and serum antibodies from infected persons to obtain a measurement matrix of binding data. Both titration curves were simultaneously fitted using an algorithm based on the generalized logistic function and adapted for analyzing thermodynamic variables of binding. We obtained equilibrium affinity constants and chemical potentials for distinct antibody classes. These variables reflect the quality and the effective quantity of serum antibodies, respectively. The proposed fluorescent dual-titration microspot immunoassay can generate truly quantitative serological data that is suitable for immunological, medical and systems biological analysis.
Inhibition of Cellular MEK/ERK Signaling Suppresses Murine Papillomavirus Type 1 Replicative Activities and Promotes Tumor Regression
Authors: Luna Adrian J. , Young Jesse M. , Sterk Rosa T. , Bondu Virginie , Schultz Fred A. , Kusewitt Donna F. , Kang Huining , Ozbun Michelle A.
Published: March 2023

Human papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for most infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expressionin vitro. Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus propertiesin vivo. We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses revealed that inhibition of MEK/ERK signaling reduces E6/E7 mRNAs, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent anti-viral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies.

Significance Statement

Persistent human papillomavirus (HPV) infections cause significant morbidity and oncogenic HPV infections can progress to anogenital and oropharyngeal cancers. Despite the availability of effective prophylactic HPV vaccines, millions of unvaccinated individuals, and those currently infected will develop HPV-related diseases over the next two decades and beyond. Thus, it remains critical to identify effective antivirals against papillomaviruses. Using a mouse papillomavirus model of HPV infection, this study reveals that cellular MEK1/2 signaling supports viral tumorigenesis. The MEK1/2 inhibitor, trametinib, demonstrates potent antiviral activities and promotes tumor regression. This work provides insight into the conserved regulation of papillomavirus gene expression by MEK1/2 signaling and reveals this cellular pathway as a promising therapeutic target for the treatment of papillomavirus diseases.
Genome-wide methylome stability and parental effects in the worldwide distributed Lombardy poplar
Authors: Broeck An Vanden , Meese Tim , Verschelde Pieter , Cox Karen , Heinze Berthold , Deforce Dieter , Meester Ellen De , Nieuwerburgh Filip Van
Published: March 2023

Background

Despite the increasing number of epigenomic studies in plants, little is known about the forces that shape the methylome in long-lived woody perennials. The Lombardy poplar offers an ideal opportunity to investigate the impact of the individual environmental history of trees on the methylome.

Results

We present the results of three interconnected experiments on Lombardy poplar. In the first experiment, we investigated methylome variability during a growing season and across vegetatively reproduced generations. We found that ramets collected over Europe and raised in common conditions have stable methylomes in symmetrical CG-contexts. In contrast, seasonal dynamics occurred in methylation patterns in CHH-context. In the second experiment, we investigated whether methylome patterns of plants grown in a non-parental environment correlate with the parental climate. We did not observe a biological relevant pattern that significantly correlates with the parental climate. Finally, we investigated whether the parental environment has persistent carry-over effects on the vegetative offsprings’ phenotype. We combined new bud set observations of three consecutive growing seasons with former published bud set data. Using a linear mixed effects analysis, we found a statistically significant but weak short-term, parental carry-over effect on the timing of bud set. However, this effect was negligible compared to the direct effects of the offspring environment.

Conclusions

Genome-wide cytosine methylation patterns in symmetrical CG-context are stable in Lombardy poplar and appear to be mainly the result of random processes. In this widespread poplar clone, methylation patterns in CG-context can be used as bio-markers to infer a common ancestor and thus to investigate the recent environmental history of a specific Lombardy poplar. The Lombardy poplar shows high phenotypic plasticity in a novel environment which enabled this clonal tree to adapt and survive all over the temperate regions of the world.
Swimming, Feeding and Inversion of Multicellular Choanoflagellate Sheets
Authors: Fung Lloyd , Konkol Adam , Ishikawa Takuji , Larson Ben T. , Brunet Thibaut , Goldstein Raymond E.
Published: March 2023

The recent discovery of the striking sheet-like multicellular choanoflagellate speciesChoanoeca flexathat dynamically interconverts between two hemispherical forms of opposite orientation raises fundamental questions in cell and evolutionary biology, as choanoflagellates are the closest living relatives of animals. It similarly motivates questions in fluid and solid mechanics concerning the differential swimming speeds in the two states and the mechanism of curvature inversion triggered by changes in the geometry of microvilli emanating from each cell. Here we develop fluid dynamical and mechanical models to address these observations and show that they capture the main features of the swimming, feeding, and inversion ofC. flexacolonies.
Using a handful of transcriptomes to detect sex-linked markers in a lizard with homomorphic sex chromosomes
Authors: Saunders Paul A. , Ferre-Ortega Carles , Hill Peta , Simakov Oleg , Ezaz Tariq , Burridge Christopher P. , Wapstra Erik
Published: March 2023

To understand the biology of a species it is often crucial to be able to differentiate males and females. Many species lack distinguishable sexually dimorphic traits, but in those that possess sex chromosomes, molecular sexing offers a good alternative. Designing molecular sexing assays is typically achieved through the comparison of male and female genomic sequences, often from reduced-representation sequencing. However, in many non-model species sex chromosomes are poorly differentiated, and identifying sex-limited sequences and developing PCR-based sexing assays is challenging without additional genomic resources. Here we highlight a simple procedure for detection of sex-linked markers based on transcriptomes that circumvents limitations of other approaches. We apply it to the spotted snow skinkCarinascincus ocellatus, a lizard with homomorphic XY chromosomes that also experiences environmentally-induced sex reversal. With transcriptomes from 3 males and 3 females alone, we identify thousands of putative Y-linked sequences. We confirm linkage through alignment of assembled transcripts to a distantly related genome, and readily design PCR primers to sexC. ocellatusand related species. In addition to providing an important molecular sexing tool for these species, this approach also facilitated valuable comparisons of sex determining systems on a large taxonomic scale.
Step-wise mechanical unfolding and dissociation of the GAIN domains of ADGRG1/GPR56, ADGRL1/Latrophilin-1 and ADGRB3/BAI3: insights into the mechanical activation hypothesis of adhesion G protein-coupled receptors
Authors: Fu Chaoyu , Huang Wenmao , Tang Qingnan , Niu Minghui , Guo Shiwen , Langenhan Tobias , Song Gaojie , Yan Jie
Published: March 2023

Adhesion G protein-coupled receptors (aGPCRs) are a large family within the superfamily of G protein-coupled receptors involved in various physiological processes. One unique feature of aGPCRs is their long N-terminal extracellular regions (ECRs), which contain adhesive domains and a GPCR autoproteolysis-inducing (GAIN) domain. This GAIN domain promotes autoproteolytic cleavage of aGPCRs into N- and C-terminal fragments (NTF, CTF, respectively) after receptor biosynthesis. aGPCR signaling involves an interplay between the NTF and CTF that can be me-chanically activated or modulated. However, how force affects the conformation/structure of the GAIN domain as a central structural element in aGPCR activation remains largely unknown. In this study, we investigated the mechanical stability of the GAIN domains of three aGPCRs from subfamilies B, G and L at a loading rate of 1 pN/s. Our findings demonstrate that the GAIN domains can be destabilized by forces from a few to 20 piconewtons (pN). Specifically, for the autocleaved aGPCRs, ADGRG1/GPR56 and ADGRL1/Latrophilin-1, forces over this range can cause detachment of the GAIN domain from the membrane-proximal Stachel element, which serves as an endogenous tethered agonist to aGPCRs, typically preceded with GAIN domain unfolding. For the non-cleavable aGPCR ADGRB3/BAI3, the GAIN domain undergoes complex mechanical unfolding over a similar force range. We also demonstrate that detachment of the GAIN domain can take place during cell migration, provided that the linkage between aGPCR and extracellular matrix is sufficiently stable. These results suggest that both structural stability of the GAIN domain and NTF/CTF dissociation are sensitive to physiological ranges of tensile forces, providing insights into the mechanical activation hypothesis of aGPCRs.
FASN-deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress
Authors: Dai Wenting , Wang Zhichao , Wang Guan , Wang Qiong A. , DeBerardinis Ralph , Jiang Lei
Published: March 2023

Fatty acid synthase (FASN) maintainsde novolipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-CoA is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation under hypoxia. Here we show that reductive carboxylation also occurs in the absence of DNL in cells with defective FASN. In this state, reductive carboxylation was mainly catalyzed by isocitrate dehydrogenase-1 (IDH1) in the cytosol, but IDH1-generated citrate was not used for DNL. Metabolic flux analysis (MFA) revealed that FASN-deficiency induced a net cytosol-to-mitochondria citrate flux through citrate transport protein (CTP). A similar pathway was previously shown to mitigate detachment-induced mitochondrial reactive oxygen species (mtROS) in anchorage-independent tumor spheroids. We further demonstrate that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Together with the reduced FASN activity in tumor spheroids, these data indicate that anchorage-independent malignant cells trade FASN-supported rapid growth for a cytosol-to-mitochondria citrate flux to gain redox capacity against detachment-induced oxidative stress.
RAPD amplicon sequence based metagenomics analysis of microbial communities associated with clove (Syzygium aromaticum) from Kerala, India
Authors: Santhakumari Sreekala Gangappan , Santhosh Ramachandran Sarojini
Published: March 2023

Four clove clones (BRC1, BRC3 MRC5 and MRC7) were collected from different places of ponmudi hills, Kerala, India. DNA was extracted and RAPD analysis was designed to evaluate clove clonal variation. Upon analysis bacterial sequences were also identified among clove genome sequences. So the clove and microbial sequences were separated and performed an analysis to identify clove associated metagenome. The major microbes present wereMycobacterium canettii Xanthomaonas euvesicateria, KlebsiellaandPsuedomonas. A total of 88 genus of microbes were present altogether in four clones ofSyzygium aromaticum. There were few fungal species were also identified,Fusarium pseudograminearumwas the major fungus associated with all four different clones of clove.
Single-cell expression predicts neuron specific protein homeostasis networks
Authors: Pechmann Sebastian
Published: March 2023

The protein homeostasis network keeps proteins in their correct shapes and avoids unwanted protein aggregation. In turn, the accumulation of aberrantly misfolded proteins has been directly associated with the onset of aging-associated neurodegenerative diseases such as Alzheimer’s and Parkinson’s. However, a detailed and rational understanding of how protein homeostasis is achieved in health, and how it can be targeted for therapeutic intervention in diseases remains missing. Here, large-scale single-cell expression data from the Allen Brain Map is analyzed to investigate the transcription regulation of the core protein homeostasis network across the human brain. Remarkably, distinct expression profiles suggest specialized protein homeostasis networks with systematic adaptations in excitatory neurons, inhibitory neurons, and non-neuronal cells. Moreover, several chaperones and Ubiquitin ligases are found transcriptionally coregulated with genes important for synapse formation and maintenance, thus linking protein homeostasis to the regulation of neuronal function. Finally, evolutionary analyses highlight the conservation of an elevated interaction density in the chaperone network, suggesting that one of the most exciting aspects of chaperone action may yet be discovered in their collective action at the systems level. More generally, our work highlights the power of computational analyses for breaking down complexity and gaining complementary insights into fundamental biological problems.
The impact of paramutations on the invasion dynamics of transposable elements
Authors: Scarpa Almorò , Kofler Robert
Published: March 2023

According to the prevailing view, the trap model, invading transposable elements (TEs) are stopped in their proliferation when a TE copy jumps into a piRNA cluster, which triggers the emergence of piRNAs that silence the TE. One crucial component in the host defence are paramutations. Mediated by maternally deposited piRNAs, paramutations convert TE insertions into piRNA producing loci, thereby transforming selfish TEs into agents of the host defence. Despite this significant effect, the impact of paramutations on the dynamics of TE invasions remains unknown. To address this issue, we performed extensive forward simulations of TE invasions with piRNA clusters and paramutations. We found that paramutations significantly affect TE dynamics, by accelerating the silencing of TE invasions, reducing the number of insertions accumulating during the invasions and mitigating the fitness cost of TEs. We also demonstrate that piRNA production induced by paramutations, an epigenetically inherited trait, may be positively selected. Finally, we show that paramutations may account for three important open problems with the trap model. Firstly, paramutated TE insertions may compensate for the insufficient number of insertions in piRNA clusters observed in previous studies. Secondly, paramutations may explain the discrepancy between the observed and the expected abundance of different TE families inD. melanogaster. Thirdly, paramutations render piRNA clusters dispensable once the host defence has been established, which may account for the lack of TE activation when three major piRNA clusters were deleted in a previous study.
Nocturnal increase in cerebrospinal fluid secretion as a regulator of intracranial pressure
Authors: Steffensen Annette Buur , Edelbo Beatriche Louise , Barbuskaite Dagne , Andreassen Søren Norge , Olsen Markus Harboe , Møller Kirsten , MacAulay Nanna
Published: March 2023

It is crucial to maintain the intracranial pressure (ICP) within the physiological range to ensure proper brain function. The ICP may fluctuate during the light-dark phase cycle, complicating diagnosis and treatment choice in patients with pressure-related disorders. Such ICP fluctuations may originate in circadian or sleep-wake cycle-mediated modulation of cerebrospinal fluid (CSF) flow dynamics, which in addition could support diurnal regulation of brain waste clearance. Through a combination of patient data andin vivotelemetric pressure measurements in adult male rats, we demonstrated that ICP increases in the dark phase in both species, independently of vascular parameters. This increase aligns with elevated CSF collection in patients and CSF production rate in rats, the latter obtained with the ventriculo-cisternal perfusion assay. The dark-phase increase in CSF secretion in rats was, in part, assigned to increased transport activity of the choroid plexus Na+,K+,2Cl-cotransporter (NKCC1), which is implicated in CSF secretion by this tissue. In conclusion, CSF secretion, and thus ICP, increases in the dark phase in humans and rats, irrespective of their diurnal/nocturnal activity preference, in part due to altered choroid plexus transport activity. Our findings suggest that CSF dynamics are modulated by the circadian rhythm, rather than merely sleep itself.
An N-acetyltransferase required for EsxA N-terminal protein acetylation and virulence in Mycobacterium marinum.
Authors: Collars Owen A. , Jones Bradley S , Hu Daniel D , Weaver Simon D , Champion Matthew M , Champion Patricia A.
Published: March 2023

N-terminal protein acetylation is a ubiquitous post-translational modification that broadly impacts diverse cellular processes in higher organisms. Bacterial proteins are also N-terminally acetylated, but the mechanisms and consequences of this modification in bacteria are poorly understood. We previously quantified widespread N-terminal protein acetylation in pathogenic mycobacteria (C. R. Thompson, M. M. Champion, and P.A. Champion, J Proteome Res 17(9): 3246-3258, 2018, https:// doi: 10.1021/acs.jproteome.8b00373). The major virulence factor EsxA (ESAT-6, Early secreted antigen, 6kDa) was one of the first N-terminally acetylated proteins identified in bacteria. EsxA is conserved in mycobacterial pathogens, including Mycobacterium tuberculosis and Mycobacterium marinum, a non-tubercular mycobacterial species that causes tuberculosis-like disease in ectotherms. However, enzyme responsible for EsxA N-terminal acetylation has been elusive. Here, we used genetics, molecular biology, and mass-spectroscopy based proteomics to demonstrate that MMAR_1839 (renamed Emp1, ESX-1 modifying protein, 1) is the putative N-acetyl transferase (NAT) solely responsible for EsxA acetylation in Mycobacterium marinum. We demonstrated that ERD_3144, the orthologous gene in M. tuberculosis Erdman, is functionally equivalent to Emp1. We identified at least 22 additional proteins that require Emp1 for acetylation, demonstrating that this putative NAT is not dedicated to EsxA. Finally, we showed that loss of emp1 resulted in a significant reduction in the ability of M. marinum to cause macrophage cytolysis. Collectively, this study identified a NAT required for N-terminal acetylation in Mycobacterium and provided insight into the requirement of N-terminal acetylation of EsxA and other proteins in mycobacterial virulence in the macrophage.
Mapping the landscape of magnetic field effects on neural regeneration and repair: a systematic review, mathematical model, and meta-analysis
Authors: McGraw Meghan , Gilmer Gabrielle , Bergmann Juliana , Seshan Vishnu , Wang Kai , Pekker David , Modo Michel , Ambrosio Fabrisia
Published: March 2023

Although magnetic field exposure is a well-established diagnostic tool, its use as a therapeutic in regenerative medicine is relatively new. Our goal here was to evaluate how magnetic fields affect neural repairin vitroby performing a systematic review of the literature, mathematical modeling, and meta-analyses. The 38 included articles presented with high heterogeneity, representing 13 cell types, magnitudes ranging from 0.0002-10,000 mT, frequencies from 0-150 Hz, and exposure times lasting from one hour to several weeks. Mathematical modeling revealed that increasing magnetic field magnitude increases neural progenitor cell (NPC) viability. For regenerative processes that were not influenced by magnitude, frequency, and time, we integrated data with meta-analyses. Results revealed that magnetic field exposure increases NPC proliferation while decreasing astrocytic differentiation. Collectively, our work identifies neural repair processes that may be most responsive to magnetic field exposure and provides a framework for novel hypothesis and technology development.
Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygousHNF1B-associated dysplastic kidney malformations
Authors: Bantounas Ioannis , Rooney Kirsty M. , Lopes Filipa M. , Tengku Faris , Woods Steven , Zeef Leo A. H. , Kuba Shweta Y. , Bates Nicola , Hummelgaard Sandra , Hillman Katherine A , Cereghini Silvia , Woolf Adrian S. , Kimber Susan J.
Published: March 2023

Hepatocyte nuclear factor 1B(HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. HeterozygousHNF1Bmutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygousHNF1Bmutant kidney organoids from CRISPR-Cas9 gene-edited human ESCs and iPSCs reprogrammed from a family withHNF1B-asscociated DKMs. Mutant organoids contained enlarged malformed tubules and displayed deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cAMP-mediated dilatation seen in controls. Bioinformatic analyses indicated abnormal WNT, calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 was upregulated in mutant organoids and was detected in their tubules and in fetal human DKM dysplastic epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule morphogenesis and functional differentiation. They additionally suggest druggable targets to ameliorate disease.
Leveraging gene correlations in single cell transcriptomic data
Authors: Silkwood Kai , Dollinger Emmanuel , Gervin Josh , Atwood Scott , Nie Qing , Lander Arthur D.
Published: March 2023

BACKGROUND

Many approaches have been developed to overcome technical noise in single cell RNA-sequencing (scRNAseq). As researchers dig deeper into data—looking for rare cell types, subtleties of cell states, and details of gene regulatory networks—there is a growing need for algorithms with controllable accuracy and fewerad hocparameters and thresholds. Impeding this goal is the fact that an appropriate null distribution for scRNAseq cannot simply be extracted from data when ground truth about biological variation is unknown (i.e., usually).

RESULTS

We approach this problem analytically, assuming that scRNAseq data reflect only cell heterogeneity (what we seek to characterize), transcriptional noise (temporal fluctuations randomly distributed across cells), and sampling error (i.e., Poisson noise). We analyze scRNAseq data without normalization—a step that skews distributions, particularly for sparse data—and calculatep-values associated with key statistics. We develop an improved method for selecting features for cell clustering and identifying gene-gene correlations, both positive and negative. Using simulated data, we show that this method, which we call BigSur (Basic Informatics andGeneStatistics fromUnnormalizedReads), captures even weak yet significant correlation structures in scRNAseq data. Applying BigSur to data from a clonal human melanoma cell line, we identify thousands of correlations that, when clustered without supervision into gene communities, align with known cellular components and biological processes, and highlight potentially novel cell biological relationships.

CONCLUSIONS

New insights into functionally relevant gene regulatory networks can be obtained using a statistically grounded approach to the identification of gene-gene correlations.
A novel ground truth dataset enables robust 3D nuclear instance segmentation in early mouse embryos
Authors: Nunley Hayden , Shao Binglun , Grover Prateek , Singh Jaspreet , Joyce Bradley , Kim-Yip Rebecca , Kohrman Abraham , Watters Aaron , Gal Zsombor , Kickuth Alison , Chalifoux Madeleine , Shvartsman Stanislav , Posfai Eszter , Brown Lisa
Published: March 2023

For investigations into fate specification and cell rearrangements in live images of preimplantation embryos, automated and accurate 3D instance segmentation of nuclei is invaluable; however, the performance of segmentation methods is limited by the images' low signal-to-noise ratio and high voxel anisotropy and the nuclei's dense packing and variable shapes. Supervised machine learning approaches have the potential to radically improve segmentation accuracy but are hampered by a lack of fully annotated 3D data. In this work, we first establish a novel mouse line expressing near-infrared nuclear reporter H2B-miRFP720. H2B-miRFP720 is the longest wavelength nuclear reporter in mice and can be imaged simultaneously with other reporters with minimal overlap. We then generate a dataset, which we call BlastoSPIM, of 3D microscopy images of H2B-miRFP720-expressing embryos with ground truth for nuclear instance segmentation. Using BlastoSPIM, we benchmark the performance of five convolutional neural networks and identify Stardist-3D as the most accurate instance segmentation method across preimplantation development. Stardist-3D, trained on BlastoSPIM, performs robustly up to the end of preimplantation development (> 100 nuclei) and enables studies of fate patterning in the late blastocyst. We, then, demonstrate BlastoSPIM's usefulness as pre-train data for related problems. BlastoSPIM and its corresponding Stardist-3D models are available at: blastospim.flatironinstitute.org.
Quorum Sensing Modulates Bacterial Virulence and Colonization Dynamics During an Enteric Infection
Authors: Peña-Díaz Jorge , Woodward Sarah E. , Creus-Cuadros Anna , Serapio-Palacios Antonio , Deng Wanyin , Finlay B. Brett
Published: March 2023

Quorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behaviour according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered thatCitrobacter rodentium, a natural murine pathogen used to model human infection by pathogenicEscherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation bothin vitroand across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence ofC. rodentiumis enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogen’s spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.
GenoPipe: identifying the genotype of origin within (epi)genomic datasets
Authors: Lang Olivia , Srivastava Divyanshi , Pugh B. Franklin , Lai William KM
Published: March 2023

Confidence in experimental results is critical for discovery. As the scale of data generation in genomics has grown exponentially, experimental error has likely kept pace despite the best efforts of many laboratories. Technical mistakes can and do occur at nearly every stage of a genomics assay (i.e., cell line contamination, reagent swapping, tube mislabelling, etc.) and are often difficult to identify post-execution. However, the DNA sequenced in genomic experiments contains certain markers (e.g., indels) encoded within and can often be ascertained forensically from experimental datasets. We developed the Genotype validation Pipeline (GenoPipe), a suite of heuristic tools that operate together directly on raw and aligned sequencing data from individual high-throughput sequencing experiments to characterize the underlying genome of the source material. We demonstrate how GenoPipe validates and rescues erroneously annotated experiments by identifying unique markers inherent to an organism’s genome (i.e., epitope insertions, gene deletions, and SNPs).