bluuebunny/crossref_metadata_2025 huggingface dataset:
Positional information modulates transient regeneration-activated cell states during vertebrate appendage regeneration
Authors: Granillo Augusto Ortega , Zamora Daniel , Schnittker Robert R. , Scott Allison R. , Russell Jonathon , Brewster Carolyn E. , Ross Eric J. , Acheampong Daniel A. , Ferro Kevin , Morrison Jason A. , Rubinstein Boris Y. , Perera Anoja G. , Wang Wei , Alvarado Alejandro Sánchez
Published: March 2024

Injury is a common occurrence in the life of organisms. Because the extent of damage cannot be predicted, injured organisms must determine how much tissue needs to be restored. It is known that amputation position determines the regeneration speed of amputated appendages in regeneration-competent animals. Yet, it is not clear how positional information is conveyed during regeneration. Here, we investigated tissue dynamics in regenerating caudal fins in the African killifish (Nothobranchius furzeri). We report position-specific, differential modulation of the spatial distribution, duration, and magnitude of proliferation. Regenerating fins profiled by single cell RNA sequencing identified a Transient Regeneration-Activated Cell State (TRACS) that is amplified to match a given amputation position. We located this TRACS to the basal epidermis and found them to express components and modifiers of the extracellular matrix (ECM). We propose a role for these cells in transducing positional information to the regenerating blastema by remodeling the ECM.

Highlights

Amputation position changes tissue-wide proliferation response

Transcriptional compartmentalization is relative to injury type

Regeneration deploys Transient Regeneration-Activated Cell States

Prediction: positional information is transduced by ECM changes during regeneration
Soft skeletons transmit force with variable gearing
Authors: Ellers Olaf , Ellers Kai-Isaak , Johnson Amy S. , Po Theodora , Heydari Sina , Kanso Eva , McHenry Matthew J.
Published: March 2024

A hydrostatic skeleton allows a soft body to transmit muscular force via internal pressure. A human’s tongue, an octopus’ arm, and a nematode’s body illustrate the pervasive presence of hydrostatic skeletons among animals, which has inspired the design of soft engineered actuators. However, there is a need for a theoretical basis for understanding how hydrostatic skeletons apply mechanical work. We therefore model the shape change and mechanics of natural and engineered hydrostatic skeletons to determine their mechanical advantage (MA) and displacement advantage (DA). These models apply to a variety of biological structures, but we explicitly consider the tube feet of a sea star and the body segments of an earthworm, and contrast them with a hydraulic press and a McKibben actuator. A helical winding of stiff, elastic fibers around these soft actuators plays a critical role in their mechanics by maintaining a cylindrical shape, distributing forces throughout the structure, and storing elastic energy. In contrast to a single-joint lever system, soft hydrostats exhibit variable gearing with changes in MA generated by deformation in the skeleton. We found that this gearing is affected by the transmission efficiency of mechanical work (MA × DA) or, equivalently, the ratio of output to input work), which changes with the capacity to store elastic energy within helically wrapped fibers or associated musculature. This modeling offers a conceptual basis for understanding the relationship between the morphology of hydrostatic skeletons and their mechanical performance.
A Synergistic Desmin-SPARC Axis Influences Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation
Authors: Leitner Lucia , Schultheis Martina , Hofstetter Franziska , Rudolf Claudia , Kizner Valeria , Fiedler Kerstin , Konrad Marie-Therese , Höbaus Julia , Genini Marco , Kober Julia , Ableitner Elisabeth , Gmaschitz Teresa , Walder Diana , Weitzer Georg
Published: March 2024

BACKGROUND

The mammalian heart contains cardiac stem cells throughout life, but it has not been possible to harness or stimulate these cells to repair damaged myocardium in vivo. Assuming physiological relevance of these cells, which have evolved and have been maintained throughout evolution, we are investigating their function using mouse cardiac stem cell lines as an in vitro model system.

METHODS

Here we use genetically modified embryonic stem cells and cardiac stem cells from the mouse as model systems to study the influence of desmin and Secreted Protein Acidic and Rich in Cysteine (SPARC) on cardiomyogenesis in embryoid bodies and cardiac bodies. We analyze their expression in self-renewing and differentiating stem cells by fluorescence microscopy, RT-qPCR, quantitative Western blotting and fluorescence activated cell sorting, and assess their influence on the expression of myocardial transcription factors.

RESULTS

In embryoid bodies, desmin induces expression and secretion of SPARC, which promotes cardiomyogenesis. Cardiac stem cells secrete substantial amounts of SPARC, which also promotes cardiomyogenesis in a concentration-dependent, autocrine manner and promotes expression of myocardial transcription factors anddesmin. Desmin and SPARC interact genetically and form a positive feedback loop and secreted SPARC negatively influences sparc mRNA expression. Finally, SPARC rescues cardiomyogenic desmin-haploinsufficiency in cardiac stem cells in a glycosylation-dependent manner, increases the phosphorylation of Smad2 and induces the expression ofgata4, nkx2.5andmef2C.

CONCLUSIONS

Demonstration that desmin-induced autocrine secretion of SPARC in cardiac stem cells promotes cardiomyogenesis raises the possibility that a physiological function of cardiac stem cells in the adult and aging heart may be the gland-like secretion of factors such as SPARC that modulate age-related and adverse environmental influences and thereby contribute to cardiac homeostasis throughout life.
Lost Opportunity in Tobacco Cessation Care: Impact of Underbilling in a Large Health System
Authors: Baughman Derek J , Rauhut Marcus , Anselm Edward
Published: March 2024

Introduction

Tobacco cessation remains a critical challenge in healthcare, with evidence-based interventions often under-utilized due to misaligned economic incentives and inadequate training. This study aims to quantify the economic impact of missed billing opportunities for tobacco cessation in a healthcare system, thereby assessing potential revenue loss and evaluating the effectiveness of systems-based approaches in enhancing tobacco cessation efforts.

Methods

A retrospective cohort study utilized aggregated de-identified patient health data from an 8-hospital regional health system across Pennsylvania and Maryland, from 1/1/21 to 12/31/23. The analysis focused on primary care encounters eligible for tobacco cessation counseling (CPT codes 99406 or 99407), with potential revenue calculated based on the Medicare reimbursement rate.

Results

Over three years, and 507,656 office visits, only 1,557 (0.3%) of encounters with persons using tobacco were billed for cessation services. The estimated total potential revenue gained if each person who was identified as using tobacco was billed consistently for tobacco cessation counseling was $5,947,018.13, and $1,982,339.38 annually.

Conclusions

The study reveals a significant gap between the potential and actual billing for tobacco cessation services, highlighting not only the financial implications of missed opportunities but the validation of health system’s public health impact. Underbilling contributes to considerable annual revenue loss and undermines primary prevention efforts against tobacco-related diseases. Our findings illuminate the need for enhanced billing practices and systemic changes, including policy improvements that influence proper billing to promote public health benefits through improved tobacco cessation interventions.
Perinatal Impacts of Cannabis and Nicotine: An Analysis of the Cannabis Use During Development and Early Life Study
Authors: Trammel Cassandra J. , Agrawal Arpana , Bogdan Ryan , Lawlor Megan , Raghuraman Nandini , Kelly Jeannie C. , Smyser Christopher D. , Rogers Cynthia , Carter Ebony B.
Published: March 2024

OBJECTIVE

Evaluate associations between prenatal cannabis use (PCU) and perinatal outcomes.

METHODS

We performed an interval analysis of a prospective cohort study of pregnant individuals with pre-pregnancy cannabis use and negative self-report of nicotine use, comparing those who continued cannabis through pregnancy with those who stopped. Patients underwent interviews and urine drug screening for cannabis and cotinine, a nicotine metabolite, in each trimester. The primary outcome was small for gestational age (SGA) at delivery. Secondary outcomes included antenatal and postpartum complications, mode of delivery, and neonatal outcomes. Secondary analyses included stratification by intensity of cannabis use and urinary cotinine positivity.

RESULTS

Birthing persons with PCU differed in age (25.5 vs 27.8 years, p=0.001), body mass index (BMI; 27.4 vs 30.9, p=0.001), area deprivation index percentiles (92% PCU vs 88%, p=0.013), cotinine positivity (42.8% vs 10.8%, p<0.001), Hispanic ethnicity (2% vs 7.2%, p=0.009), and education attainment beyond high school (29.4% vs 50%, p<0.001) compared to controls. Birthing person outcomes did not differ. Risks of SGA and other neonatal outcomes did not differ when adjusted for confounders on initial analysis, or with stratification by intensity of cannabis use. Despite negative self-report for nicotine, 42.8% of PCU patients tested positive for cotinine (PCU+c). PCU+c was associated with increased risk of SGA and birthweight less than the 5thpercentile, compared to PCU cases without nicotine exposure (17.4% vs 8.3%, aRR 2.7 [1.21-5.38], 35.8% vs 18.6%, aRR 2.4 [1.51-3.48]), and controls (35.8% vs 12.9%, aRR 2.75 [1.64-4.13]). Cotinine-negative PCU patients and controls did not differ.

CONCLUSION

PCU was not independently associated with adverse birthing person outcomes. Many patients demonstrated nicotine exposure, either via inadvertent exposure or undisclosed use. While neonates exposed to cannabis alone did not differ from unexposed neonates, those exposed to both cannabis and nicotine were at increased risk of SGA.
Utility of quantitative measurement ofT2using Restriction Spectrum Imaging for detection of clinically significant prostate cancer
Authors: Domingo Mariluz Rojo , Conlin Christopher C , Karunamuni Roshan A , Ollison Courtney , Baxter Madison T , Kallis Karoline , Do Deondre D , Song Yuze , Kuperman Joshua M , Shabaik Ahmed S , Hahn Michael E , Murphy Paul M , Rakow-Penner Rebecca , Dale Anders M , Seibert Tyler M
Published: March 2024

Background

The Restriction Spectrum Imaging restriction score (RSIrs) has demonstrated higher diagnostic accuracy for clinically significant prostate cancer (csPCa) than conventional DWI. Both diffusion andT2properties of prostate tissue inform the RSI signal, and studies have shown that each may be valuable for csPCa discrimination.

Purpose

To determine whether prostateT2varies across RSI compartments and in the presence of csPCa, and to evaluate whether consideration of compartmentalT2(cT2) improves csPCa detection over RSIrs alone.

Study Type

Retrospective.

Population

Two cohorts (46 and 195 patients) scanned for csPCa.

Field Strength/Sequence

3T multi-b-value DWI acquired at multiple TEs.

Assessment

cT2values were computed from multi-TE RSI data and compared between RSI model compartments. csPCa detection was compared between RSIrs and a logistic regression model (LRM) for predicting the probability of csPCa using cT2in combination with RSI measurements.

Statistical Tests

Two-sample t-tests (α=0.05) were used to compare cT2values between compartments and between patients with and without csPCa. Area under the receiver operating characteristic curve (AUC) was used to evaluate csPCa detection performance.

Results

In both cohorts,T2differed (p<0.05) across all RSI compartments (C1,C2,C3,C4). Voxel-level data from cohort 1 showed thatT2differed between normal and cancerous tissue inC1,C2,C3(p<0.001). Whole-prostateT2differed between patients with and without csPCa inC3(p=0.02). In cohort 2, whole-prostateT2differed inC1(p=0.01),C3(p=0.01), andC4(p<0.01). Consideration of cT2improved csPCa discrimination compared to diffusion alone, but not compared to RSIrs [cohort 1: 0.80 vs 0.70 (diffusion) and 0.80 (RSIrs), cohort 2: 0.72 vs 0.65 (diffusion) and 0.72 (RSIrs)].

Data Conclusion

Significant differences in cT2were observed between normal and cancerous prostatic tissue. With our data, however, consideration of cT2did not significantly improve cancer detection performance over RSIrs alone.
Phenyllactic Acid is Physiologically Released from Skeletal Muscle and Contributes to the Beneficial Effects of Physical Exercise in Humans
Authors: Hoene Miriam , Zhao Xinjie , Hu Chunxiu , Birkenfeld Andreas L. , Peter Andreas , Nieß Andreas M. , Moller Anja , Li Qi , Lehmann Rainer , Plomgaard Peter , Xu Guowang , Weigert Cora
Published: March 2024

Aims/hypothesis

While physical activity is clearly beneficial in combating type 2 diabetes, the underlying molecular mechanisms are incompletely understood. Moreover, there is a considerable degree of variability in the individual response to exercise-based lifestyle interventions that remains to be explained. We aimed to identify novel exercise-induced metabolites that could mediate the improvement in glycemic control and reduction of obesity and contribute to individual differences in the response to exercise interventions.

Methods

We studied acute exercise- and training-induced changes in plasma metabolites in sedentary subjects with overweight (8 male, 14 female) participating in an eight-week supervised training program flanked by two acute endurance exercise sessions. Plasma metabolites were quantified using LC- and CE-MS. In a separate study (n=9 lean males), we assessed metabolite fluxes over the leg using arterial and venous catheters. Functional analyses were performed in primary blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS) or the saturated fatty acid palmitate.

Results

The amino acid breakdown products 3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid and indolelactic acid were increased after both acute exercise and training. All three aromatic lactic acids, which so far mainly received attention as bacterial metabolites, exhibited an efflux from the leg. PLA showed the largest increase after both acute exercise and training, of 57% and 20% respectively. The magnitude of the acute exercise-induced increase in PLA correlated with a decrease in subcutaneous adipose tissue volume and an improvement in insulin sensitivity over the course of the intervention. Furthermore, both isomers, D- and L-PLA, counteracted inflammatory cytokine production in PBMCs.

Conclusions/interpretation

Our findings indicate that PLA is physiologically released from skeletal muscle and can contribute to the anti-inflammatory effects of exercise as well as to individual difference in the response to lifestyle interventions in humans. PLA and potentially, aromatic lactic acids in general may be particularly relevant metabolic regulators because they can be produced both endogenously and by the microbiome.

Trial registration

ClinicalTrials.govNCT03151590
"Everything created by a white man is for pagans": Understanding the Barriers to Childhood Immunization in North-Eastern and North-Western Nigeria.
Authors: Etim Eno-Obong , Odiachi Angela , Dougherty Leanne , Alabi Matthew A , Adetunji Adetayo , Adedimeji Adebola
Published: March 2024

Background: Despite investments by the Nigerian government and international organizations in childhood immunization to combat child mortality, coverage in many northern states remains below the national average, thereby increasing the risk of vaccine-preventable diseases. This study describes the barriers to immunization in six states in Northeast and Northwest Nigeria, which have the lowest vaccination coverage rates in the country. Method: The socioecological framework informed the design of focus group discussions that we conducted with 93 caregivers and 91 community influencers from March to July 2022 to understand their perceptions of the barriers to immunization uptake. We thematically analyzed data that were inductively and deductively coded in NVivo version 12 software. Results: Supply-side service delivery challenges were the main systems-level barriers to vaccine uptake. These included poor provider attitude, shortage of health workers, vaccine stock-out, long wait- times, and inequitable service delivery. Other barriers were, rumors, misconceptions, vaccine distrust, socio-cultural and religious norms at the community level, and spousal and family disapproval at the interpersonal level. Limited autonomy and mobility, poor vaccine knowledge and awareness of immunization benefits, and concerns about side effects were important individual-level barriers. Conclusion: Strategies for improving immunization uptake should include intensifying community sensitization, improving the numbers and quality of the health workforce, involving men and traditional institutions through context-appropriate channels, and addressing socio-cultural norms and concerns about adverse events after immunization.
Brugada ECG detection with self-supervised VICReg pre-training: a novel deep learning approach for rare cardiac diseases
Authors: Ronan Robert , Tarabanis Constantine , Chinitz Larry , Jankelson Lior
Published: March 2024

Existing deep learning algorithms for electrocardiogram (ECG) classification rely on supervised training approaches requiring large volumes of reliably labeled data. This limits their applicability to rare cardiac diseases like Brugada syndrome (BrS), often lacking accurately labeled ECG examples. To address labeled data constraints and the resulting limitations of supervised training approaches, we developed a novel deep learning model for BrS ECG classification using the Variance-Invariance-Covariance Regularization (VICReg) architecture for self-supervised pre-training. The VICReg model outperformed a state-of-the-art neural network in all calculated metrics, achieving an area under the receiver operating and precision-recall curves of 0.88 and 0.82, respectively. We used the VICReg model to identify missed BrS cases and hence refine the previously underestimated institutional BrS prevalence and patient outcomes. Our results provide a novel approach to rare cardiac disease identification and challenge existing BrS prevalence estimates offering a framework for other rare cardiac conditions.
WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTINGCRYPTOSPORIDIUMspp.
Authors: Bayona-Vásquez NJ , Sullivan AH , Beaudry MS , Khan A , Baptista RP , Petersen KN , Bhuiyan MIU , Brunelle B , Robinson G , Chalmers RM , Alves-Ferreira EVC , Grigg ME , AlvesFerreira Kissinger JC , Glenn TC
Published: March 2024

Cryptosporidiumspp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pureCryptosporidiumDNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infectingCryptosporidiumspp. (C. cuniculus,C. hominis,C. meleagridis,C. parvum,C. tyzzeri, andC. viatorum) to enrichCryptosporidiumspp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to targetCryptosporidiumreferences in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.
Growth patterns of caudal fin rays are informed by both external signals from the regenerating organ and remembered identity autonomous to the local tissue
Authors: Autumn Melody , Hu Yinan , Zeng Jenny , McMenamin Sarah K.
Published: March 2024

Regenerating tissues must remember or interpret their spatial position, using this information to restore original size and patterning. The external skeleton of the zebrafish caudal fin is composed of 18 rays; after any portion of the fin is amputated, position-dependent regenerative growth restores each ray to its original length. We tested for transcriptional differences during regeneration of proximal versus distal tissues and identified 489 genes that differed in proximodistal expression. Thyroid hormone directs multiple aspects of ray patterning along the proximodistal axis, and we identified 364 transcripts showing a proximodistal expression pattern that was dependent on thyroid hormone context. To test what aspects of ray positional identity are directed by extrinsic cues versus remembered identity autonomous to the tissue itself, we transplanted distal portions of rays to proximal environments and evaluated regeneration within the new location. While neighboring proximal tissue showed robust expression ofscpp7, a transcript with thyroid-regulated proximal enrichment, regenerating rays originating from transplanted distal tissue showed reduced (distal-like) expression during outgrowth. These distal-to-proximal transplants regenerated far beyond the length of the graft itself, indicating that cues from the proximal environment promoted additional growth. Nonetheless, these transplants initially regenerated at a much slower rate compared to controls, suggesting memory of distal identity was retained by the transplanted tissue. This early growth retardation caused rays that originated from transplants to become noticeably shorter than their native neighboring rays. While several aspects of fin ray morphology (bifurcation, segment length) were found to be determined by the environment, regeneration speed and ray length are remembered autonomously by tissues, persisting across multiple rounds of amputation and regeneration.

GRAPHICAL ABSTRACT
Ribonuclease Inhibitor and Angiogenin collaboratively regulate cell-type-specific global translation
Authors: Stillinovic Martina , Sarangdhar Mayuresh Anant , Andina Nicola , Tardivel Aubry , Greub Frédéric , Bombaci Giuseppe , Ansermet Camille , Heller Manfred , Keogh Adrian , Keller Irene , Angelillo-Scherrer Anne , Allam Ramanjaneyulu
Published: March 2024

Translation of mRNAs is a fundamental process that occurs in all cell-types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell-type-specific translation1–3. Despite this, it remains unclear whether global translation is controlled in a cell-type-specific manner. Here we report that a ribosome-associated protein ribonuclease inhibitor-1 (RNH1) and its binding partner Angiogenin (ANG) collaboratively regulates cell-type-specific global translation. By employing human cell-lines and mouse models, we found that deletion of RNH1 decreases global translation selectively in hematopoietic origin cells but not in the non-hematopoietic origin cells. RNH1 mediated such cell-type-specific translation is mechanistically linked to ANG. We found that ANG, which is known to regulate ribosomal biogenesis4, is predominantly expressed in non-hematopoietic origin cells and absent in hematopoietic origin cells. ANG safeguards the non-hematopoietic origin cells from RNH1-knockout-mediated translation defects by upregulating ribosomal biogenesis. Further, we discovered that RNH1 controls the translation of ribosomal protein (RP) transcripts and influences mRNA circularization. Collectively, this study unravels the existence of cell-type-specific global translation regulators and highlights the complex translation regulation in vertebrates.
A Genome-wide CRISPR screen unveils the endosomal maturation protein WDR91 as a promoter of productive ASO activity in melanoma
Authors: Menchon Grégory , Gaci Aris , Matvere Antti , Aubry Marc , Bore Aurélien , Gilot David , Goyenvalle Aurélie , Pedeux Rémy
Published: March 2024

Antisense oligonucleotides (ASOs) belong to promising therapeutics for the treatment of neurologic, muscular and metabolic disorders. Several ASOs have been approved so far and more than a hundred clinical trials are currently underway covering a dozen therapeutic areas. Yet, the mechanisms of internalization and cell trafficking of these molecules remain poorly understood. Moreover, with only a small fraction of ASOs reaching the correct cellular compartment following systemic delivery, the majority of targeted diseases requires recurrent injections of ASOs. A deeper understanding of these mechanisms would guide the improvement of their potency and thus, reduce the amount of delivered ASOs and their potential side-effects. Here, using a CRISPR screen, we investigated intracellular proteins involved in ASOs efficiency using a whole genome approach and identified several potential regulators which could significantly impact ASOs potency in melanoma cells. We validated WD Repeat Domain 91 (WDR91), a regulator of endosomal maturation, as a modulator whose depletion significantly inhibits ASO productive activity. This study provides the first list of ASO modulators using a biologically relevant assay to estimate the role of these proteins. In conclusion, these data could lead to a better understanding of the mechanisms favoring productive uptake or improved endosomal escape of ASOs.
GABAergic interneurons contribute to the fatal seizure phenotype of CLN2 disease mice
Authors: Takahashi Keigo , Rensing Nicholas R. , Eultgen Elizabeth M. , Wang Sophie H. , Nelvagal Hemanth R. , Le Steven Q. , Roberts Marie S. , Doray Balraj , Han Edward B. , Dickson Patricia I. , Wong Michael , Sands Mark S. , Cooper Jonathan D.
Published: March 2024

GABAergic interneuron deficits have been implicated in the epileptogenesis of multiple neurological diseases. While epileptic seizures are a key clinical hallmark of CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), the etiology of these seizures remains elusive. Given thatCln2R207X/R207Xmice display fatal spontaneous seizures and an early loss of several cortical interneuron populations, we hypothesized that those two events might be causally related. To address this hypothesis, we first generated an inducible transgenic mouse expressing lysosomal membrane-tethered TPP1 (TPP1LAMP1) on theCln2R207X/R207Xgenetic background to study the cell-autonomous effects of cell-type-specific TPP1 deficiency. We crossed the TPP1LAMP1 mice withVgat-Cremice to introduce interneuron-specific TPP1 deficiency.Vgat-Cre; TPP1LAMP1 mice displayed storage material accumulation in several interneuron populations both in cortex and striatum, and increased susceptibility to die after PTZ-induced seizures. Secondly, to test the role of GABAergic interneuron activity in seizure progression, we selectively activated these cells inCln2R207X/R207Xmice using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) in inVgat-Cre:Cln2R207X/R207Xmice. EEG monitoring revealed that DREADD-mediated activation of interneurons via chronic deschloroclozapine administration accelerated the onset of spontaneous seizures and seizure-associated death inVgat-Cre:Cln2R207X/R207Xmice, suggesting that modulating interneuron activity can exert influence over epileptiform abnormalities in CLN2 disease. Taken together, these results provide new mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.
Sensitive Fluorescent Biosensor Reveals Differential Subcellular Regulation of PKC
Authors: Su Qi , Zhang Jing , Lin Wei , Zhang Jin-Fan , Newton Alexandra C. , Mehta Sohum , Yang Jing , Zhang Jin
Published: March 2024

The protein kinase C (PKC) family of serine/threonine kinases, which consist of three distinctly regulated subfamilies, have long been established as critical for a variety of cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs such as the ER, lysosome, and Par signaling complexes, is unclear. Here, we present a sensitive Excitation Ratiometric (ExRai) C Kinase Activity Reporter (ExRai-CKAR2) that enables the detection of minute changes in subcellular PKC activity. Using ExRai-CKAR2 in conjunction with an enhanced diacylglycerol (DAG) biosensor capable of detecting intracellular DAG dynamics, we uncover the differential regulation of PKC isoforms at distinct subcellular locations. We find that G-protein coupled receptor (GPCR) stimulation triggers sustained PKC activity at the ER and lysosomes, primarily mediated by Ca2+sensitive conventional PKC (cPKC) and novel PKC (nPKC), respectively, with nPKC showing high basal activity due to elevated basal DAG levels on lysosome membranes. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or Par-complexes, further enabled us to detect previously inaccessible endogenous atypical PKC (aPKC) activity in 3D organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.
GTRpmix: A linked general-time reversible model for profile mixture models
Authors: Banos Hector , Wong Thomas KF. , Daneau Justin , Susko Edward , Minh Bui Quang , Lanfear Robert , Brown Matthew W. , Eme Laura , Roger Andrew J.
Published: March 2024

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common set of amino acid exchange rates (a matrix of exchangeabilities) and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile.

Currently, empirically estimated exchangeability matrices (e.g., the LG or WAG matrices) are widely used for phylogenetic inference under profile mixture models. However, such matrices were originally estimated using site homogeneous models with a single set of equilibrium amino acid frequencies; therefore unlikely to be optimal for site heterogeneous profile mixture models. Here we describe the GTRpmix model, implemented in IQ-TREE2, that allows maximum likelihood estimation of a common set of exchangeabilities for all site classes under any profile mixture model. We show that exchangeability matrices estimated in the presence of a site-heterogeneous profile mixture model differ markedly from the widely used LG matrix and dramatically improve model fit and topological estimation accuracy for empirical test cases.

Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic supermatrices under the C60 profile mixture model that can be used as fixed matrices for phylogenetic analyses. One of these, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices when combined with profile mixture models fit data much better and have improved topology estimation relative to the empirical LG matrix combined with the same underlying mixture models. Version v2.3.1 of IQ-TREE2 implementing these models is available atwww.iqtree.org.
Molecular determinants of Neu5Ac binding to a tripartite ATP independent periplasmic (TRAP) transporter
Authors: Goyal Parveen , Dhanabalan KanagaVijayan , Scalise Mariafrancesca , Friemann Rosmarie , Indiveri Cesare , Dobson Renwick C.J. , Vinothkumar Kutti R. , Ramaswamy Subramanian
Published: March 2024

N-Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino-sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of theHaemophilus influenzaeTRAP transport system, (Currieet al.,2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures ofFusobacterium nucleatumSiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na+binding sites. Two of the Na+-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.
Cell fixation improves performance ofin situcrosslinking mass spectrometry while preserving cellular ultrastructure
Authors: Michael Andrew R.M. , Amaral Bruno C. , Ball Kallie L. , Eiriksson Kristen H. , Schriemer David C.
Published: March 2024

Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, currentin vivoXL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions forin situXL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.
Novel extracellular matrix architecture on excitatory neurons revealed by HaloTag-HAPLN1
Authors: Sterin Igal , Niazi Ava , Kim Jennifer , Park Joosang , Park Sungjin
Published: March 2024

The brain’s extracellular matrix (ECM) regulates neuronal plasticity and animal behavior. ECM staining shows an aggregated pattern in a net-like structure around a subset of neurons and diffuse staining in the interstitial matrix. However, understanding the structural features of ECM deposition across various neuronal types and subcellular compartments remains limited. To visualize the organization pattern and assembly process of the hyaluronan-scaffolded ECM in the brain, we fused a HaloTag to HAPLN1, which links hyaluronan and proteoglycans. Expression or application of the probe enables us to identify spatial and temporal regulation of ECM deposition and heterogeneity in ECM aggregation among neuronal populations. Dual-color birthdating shows the ECM assembly process in culture andin vivo.Sparse expression in vivo reveals novel forms of ECM architecture around excitatory neurons and developmentally regulated dendritic ECM. Overall, our study uncovers extensive structural features of the brain’ ECM, suggesting diverse roles in regulating neuronal plasticity.
A spectral framework to map QTLs affecting joint differential networks of gene co-expression
Authors: Hu Jiaxin , Weber Jesse N. , Fuess Lauren E. , Steinel Natalie C. , Bolnick Daniel I. , Wang Miaoyan
Published: March 2024

Studying the mechanisms underlying the genotype-phenotype association is crucial in genetics. Gene expression studies have deepened our understanding of the genotype → expression → phenotype mechanisms. However, traditional expression quantitative trait loci (eQTL) methods often overlook the critical role of gene co-expression networks in translating genotype into phenotype. This gap highlights the need for more powerful statistical methods to analyze genotype → network → phenotype mechanism. Here, we develop a network-based method, called snQTL, to map quantitative trait loci affecting gene co-expression networks. Our approach tests the association between genotypes and joint differential networks of gene co-expression via a tensor-based spectral statistics, thereby overcoming the ubiquitous multiple testing challenges in existing methods. We demonstrate the effectiveness of snQTL in the analysis of three-spined stickleback (Gasterosteus aculeatus) data. Compared to conventional methods, our method snQTL uncovers chromosomal regions affecting gene co-expression networks, including one strong candidate gene that would have been missed by traditional eQTL analyses. Our framework suggests the limitation of current approaches and offers a powerful network-based tool for functional loci discoveries.

Significance statement

This work addresses a key gap in understanding the mechanistic foundations for genotype-phenotype associations. While existing expression quantitative trait loci (eQTL) methods identify candidate loci affecting gene expression variants, they often neglect the crucial role of gene co-expression networks. Here, we develop a network-based QTL framework to map genetic loci affecting the gene co-expression network. Utilizing a tensor-based spectral approach, our snQTL method estimates the differential co-expression patterns and effectively identifies the associated genetic loci. Application of snQTL to three-spined sticklebacks revealed candidate loci missed by standard methods. This work suggests the limitations of current approaches and highlights the potential of network-based functional loci discovery.
Unravelling differential Hes1 dynamics during axis elongation of mouse embryos through single-cell tracking
Authors: el Azhar Yasmine , Schulthess Pascal , van Oostrom Marek J. , Meijer Wilke H.M. , Thomas Wouter M. , Bauer Marianne , Sonnen Katharina F.
Published: March 2024

The intricate dynamics of Hes expression across diverse cell types in the developing vertebrate embryonic tail have remained elusive. To address this, we developed an endogenously tagged Hes1-Achilles mouse line, enabling precise quantification of dynamics at the single-cell resolution across various tissues. Our findings reveal striking disparities in Hes1 dynamics between presomitic mesoderm (PSM) and preneural tube (pre-NT) cells. While pre-NT cells display variable, low-amplitude oscillations, PSM cells exhibit synchronized, high-amplitude oscillations. Upon the induction of differentiation, the oscillation amplitude increases in pre-NT cells. Additionally, our study of Notch inhibition on Hes1 oscillations unveiled distinct responses in PSM and pre-NT cells, corresponding to differential Notch ligand expression dynamics. These findings suggest the involvement of separate mechanisms driving Hes1 oscillations. Thus, Hes1 demonstrates dynamic behaviour across adjacent tissues of the embryonic tail, yet the varying oscillation parameters give rise to differences in the information that can be conveyed by these dynamics.
The TTLL10 polyglycylase is stimulated by tubulin glutamylation and inhibited by polyglycylation
Authors: Cummings Steven W. , Li Yan , Spector Jeffrey O. , Kim Christopher , Roll-Mecak Antonina
Published: March 2024

ABSTRACT

Microtubules in cells have complex and developmentally stereotyped posttranslational modifications that support diverse processes such as cell division, ciliary growth and axonal specification. Glycylation, the addition of glycines, singly (monoglycylation) or in chains (polyglycylation), is primarily found on axonemal microtubules where it functions in cilia maintenance and motility. It is catalyzed by three enzymes in the tubulin tyrosine ligase- like family, TTLL3, 8 and 10. We show that TTLL8 monoglycylates both α- and β-tubulin, unlike TTLL3 which prefers β-tubulin. Microscopy and mass spectrometry show that TTLL10 requires monoglycylation for high affinity microtubule binding and elongates polyglycine chains only from pre-existing glycine branches. Surprisingly, tubulin polyglycylation inhibits TTLL10 recruitment to microtubules proportional with the number of posttranslationally added glycines, suggesting an autonomous mechanism for polyglycine chain length control. In contrast, tubulin glutamylation, which developmentally precedes polyglycylation in cilia, increases TTLL10 recruitment to microtubules, suggesting a mechanism for sequential deposition of tubulin modifications on axonemes. Our work sheds light on how the tubulin code is written by establishing the substrate preference and regulation of TTLL glycylases, and provides a minimal system for generating differentially glycylated microtubules forin vitroanalyses of the tubulin code.
Laue microdiffraction on polycrystalline samples above 1500 K achieved with the QMAX-µLaue furnace
Authors: Purushottam Raj Purohit Ravi Raj Purohit , Fowan Daniel , Arnaud Stephan , Blanc Nils , Micha Jean-Sébastien , Guinebretière René , Castelnau Olivier
Published: March 2024

X-ray Laue microdiffraction aims to characterize microstructural and mechanical fields in polycrystalline specimens at the sub-micrometre scale with a strain resolution of ∼10−4. Here, a new and unique Laue microdiffraction setup and alignment procedure is presented, allowing measurements at temperatures as high as 1500 K, with the objective to extend the technique for the study of crystalline phase transitions and associated strain-field evolution that occur at high temperatures. A method is provided to measure the real temperature encountered by the specimen, which can be critical for precise phase-transition studies, as well as a strategy to calibrate the setup geometry to account for the sample and furnace dilation using a standard α-alumina single crystal. A first application to phase transitions in a polycrystalline specimen of pure zirconia is provided as an illustrative example.
Immersive virtual reality in experiential learning for architecture design education: an action research
Authors: Ummihusna Annisa , Zairul Mohd , Ab Jalil Habibah , Sulaiman Puteri Suhaiza
Published: March 2024

Purpose

Challenges of conducting site visit activities, a vital component of architecture learning during the recent pandemic have proved our unreadiness in facing the digital future. The lack of understanding of learning technology has affected the education experience. Thus, there is a need to investigate immersive learning technology such as immersive virtual reality (IVR) to replace students’ concrete experience in the current learning setting. This study aims to answer: (1) What is the influence of IVR in experiential learning (EL) in enhancing the personal spatial experience? (2) Does IVR in EL influence students' approach to learning during the architecture design process?

Design/methodology/approach

The research was conducted as an action research design approach. Action research was employed in the first-year architecture design studio by the lecturer as a practitioner-researcher. The personal spatial experience survey was performed in the earlier phase to identify the students’ prior spatial experience. Architectural Spatial Experience Simulation (ASES) a learning tool was implemented and assessed with Architecture Design Learning Assessment (ADLA) rubric, which was developed to evaluate EL and student’s approach to learning during the architecture design learning process.

Findings

The outcomes revealed that ASES as a learning tool in EL could improve the participants’ spatial experience, particularly those with minimal prior personal spatial experience. ASES was recognized to enhance the participants’ EL experience and encourage changes in student’s approach to learning from surface to deep learning.

Originality/value

This research benefits the architecture design learning process by offering a learning tool and a framework to resolve challenges in performing site visit activities and digital learning. It also contributes by expanding the EL theory and students’ approach to learning knowledge in the architecture education field.
Getting by with a little help from friends and family: mediation model between loneliness and depressive symptoms amongst Portuguese students
Authors: Kulari Genta
Published: March 2024

Purpose

The purpose of this study is to analyse the mediating effect of friends and family as sources of perceived social support in the relationship between loneliness and depressive symptoms.

Design/methodology/approach

Survey data sample consisted of 733 university students from January to May 2023. Participants completed the UCLA loneliness scales, Multidimensional Scale of Perceived Social Support (MPSS) and Depression, Anxiety and Stress Scale (DASS-21). The SPSS programme with PROCESS macro (Model 6) was used to test the hypothesis regarding the mediation effect.

Findings

The bootstrap analysis found that friends as a source of social support mediated the relationship between loneliness and depressive symptoms. Similarly, loneliness had a significant indirect effect on depressive symptoms through the mediation of family as a source of social support. Moreover, it was found that the relationships of friends and family as sources of social support mediated the association of the aforementioned variables.

Originality/value

This research advances our understanding of social support sources from friends and family amongst university students whilst providing suggestions for interventions tackling loneliness and depressive symptoms in a university setting.