bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of reproductive isolation in a long-term evolution experiment withDrosophila melanogaster: 30 years of divergent life history selection
Authors: Robinson Chloe E , Thyagarajan Harshavardhan , Chippindale Adam K
Published: February 2023

We ask if three decades and over 1 500 generations of divergent life history selection on age at reproduction has resulted in the evolution of reproductive isolation (RI) between laboratory populations ofDrosophila melanogaster. We tested for premating, postmating-prezygotic and postzygotic reproductive isolation between 3 replicate population pairs. Large evolved differences in body size between selection treatments suggested the potential for prezygotic barriers driven by sexual selection or physical incompatibilities between the sexes. Although a simple prediction would be preference for larger size, creating directional isolation, our results from individual mate choice trials indicate that populations from both selection treatments show a marked bias towards homotypic mate choice; indicative of prezygotic RI driven by sexual selection or sexual conflict. Hybridization between the focal populations resulted in the production of viable adult flies with intermediate size and developmental traits. We observed a suggestive but statistically non-significant trend of fitness decline in the F2 generation of hybrids, but no significant evidence suggesting the evolution of postmating-prezygotic or postzygotic RI. Our findings are in accord with extant literature that posits that premating RI evolves before postmating forms of RI.
Red-purple Andean potato polyphenols have an anti-neuroblastoma effectin vitrovia apoptosis through mitochondria
Authors: Silveyra María Ximena , Andreu Adriana Balbina
Published: February 2023

Andean potatoes (Solanum tuberosumssp.andigena) are a good source of dietary polyphenols, such as phenolic acid and flavonoids. These polyphenols have several beneficial effects on human health due to their antioxidant properties. Previously, we demonstrated that polyphenol extracts from Andean potato tubers exerted a concentration-dependent cytotoxic effect in human neuroblastoma cells. However, the mechanisms involved in this cytotoxic activity were not explored. Here we show that Santa María tuber’s polyphenols activated a programmed cell death by caspase-independent apoptosis. We found that polyphenols induced cell and nucleus morphology changes and slightly affected the cell cycle. Furthermore, the polyphenols altered the neuroblastoma cells’ homeostasis redox and mitochondrial function, increasing the levels of apoptotic cells. Finally, we showed that neither Bcl-2 nor caspase-3 was involved in this mechanism of death. Our results confirmed that Santa María tuber’s polyphenols are bioactive compounds with mitochondria as a target and contribute to revalorizing Andean potatoes as a functional food. These findings demonstrated that they would be a good source of anti-tumor compounds that would induce tumor cell death even in apoptotic-resistant tumors, opening new therapeutic avenues.
EMBER multi-dimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains
Authors: Yang Hyunjun , Yuan Peng , Wu Yibing , Shi Marie , Caro Christoffer D. , Tengeiji Atsushi , Yamanoi Shigeo , Inoue Masahiro , DeGrado William F. , Condello Carlo
Published: February 2023

In neurodegenerative diseases proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformationsin situ. Here we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid binding dyes to identify six distinct different conformational strainsin vitro, as well as Aβ deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aβ and tau deposits from Down syndrome, sporadic and familial Alzheimer’s disease human brain slices. EMBER hasin situidentified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick’s disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.

Significance

In neurodegenerative diseases proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. There is a need to rapidly identify these amyloid conformationsin situ. Here we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid binding dyes to identify six distinct different conformational strainsin vitro, as well as Aβ deposits in different transgenic mouse models. Our imaging method rapidly identifies conformational differences in Aβ and tau deposits from Down syndrome, sporadic and familial Alzheimer’s disease human brain slices. We also identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick’s disease. These findings will facilitate the identification of pathogenic protein aggregates to guide research and treatment of protein misfolding diseases.
Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array
Authors: Samimi Kayvan , Desa Danielle E. , Lin Wei , Weiss Kurt , Li Joe , Huisken Jan , Miskolci Veronika , Huttenlocher Anna , Chacko Jenu V. , Velten Andreas , Rogers Jeremy D. , Eliceiri Kevin W. , Skala Melissa C.
Published: February 2023

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, andin vivoNAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.
An electrochemical approach for rapid, sensitive, and selective detection of dynorphin
Authors: Conway Sineadh M. , Kuo Chao-Cheng , Gardiner Woodrow , Wu Rui-Ni , Thang Loc V. , Gereau Graydon B. , Cirrito John R. , Yuede Carla M. , McCall Jordan G. , Al-Hasani Ream
Published: February 2023

The endogenous opioid peptide systems are critical for analgesia, reward processing, and affect, but research on their release dynamics and function has been challenging. Here, we have developed microimmunoelectrodes (MIEs) for the electrochemical detection of opioid peptides using square-wave voltammetry. Briefly, a voltage is applied to the electrode to cause oxidation of the tyrosine residue on the opioid peptide of interest, which is detected as current. To provide selectivity to these voltammetric measurements, the carbon fiber surface of the MIE is coated with an antiserum selective to the opioid peptide of interest. To test the sensitivity of the MIEs, electrodes are immersed in solutions containing different concentrations of opioid peptides, and peak oxidative current is measured. We show that dynorphin antiserum-coated electrodes are sensitive to increasing concentrations of dynorphin in the attomolar range. To confirm selectivity, we also measured the oxidative current from exposure to tyrosine and other opioid peptides in solution. Our data show that dynorphin antiserum-coated MIEs are sensitive and selective for dynorphin with little to no oxidative current observed in met-enkephalin and tyrosine solutions. Additionally, we demonstrate the utility of these MIEs in anin vitrobrain slice preparation using bath application of dynorphin as well as optogenetic activation of dynorphin release. Future work aims to use MIEsin vivofor real-time, rapid detection of endogenous opioid peptide release in awake, behaving animals.
Optimizing anesthesia and delivery approaches for dosing into lungs of mice
Authors: Seo Yurim , Qiu Longhui , Magnen Mélia , Conrad Catharina , Moussavi-Harami S. Farshid , Looney Mark R , Cleary Simon J
Published: February 2023

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in models of viral and bacterial pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus orPseudomonas aeruginosadeveloping more robust lung inflammation responses relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. Use of either of these more precise dosing methods yielded greater experimental power in the bacterial pneumonia model relative to intranasal infection. Both anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors affect experimental power and so should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.

Graphical abstract
Computational evaluation of light propagation in cylindrical bioreactors for optogenetic mammalian cell cultures
Authors: Minami Shiaki A. , Shah Priya S.
Published: February 2023

Optogenetic control of cellular pathways and gene circuits in mammalian cells is a new frontier in mammalian genetic engineering. As a low-cost, tunable, and reversible input, light is highly adept at spatiotemporal, orthogonal regulation of cellular behavior. However, light is absorbed and scattered as it travels through media and cells, and the applicability of optogenetics in larger mammalian bioreactors has not been determined. In this work, we computationally explore the size limit to which optogenetics can be applied in cylindrical bioreactors at relevant height-to-diameter ratios for mammalian cell culture. We model the propagation of light using the radiative transfer equation and consider changes in reactor volume, absorption coefficient, scattering coefficient, and scattering anisotropy. We observed sufficient light penetration for activation for bioreactor sizes of up to 80,000 L with maximal cell densities, with decreasing efficiency for larger bioreactors. For a 100,000 L bioreactor, we determined that lower cell densities of up to 1.5·107cells/mL can be supported. We conclude that optogenetics can be applied to bioreactors at an industrial scale and may be a valuable tool for specific biomanufacturing applications.
Identifying Human Specific Adverse Outcome Pathways of Per- and Polyfluoroalkyl Substances Using Liver-Chimeric Humanized Mice
Authors: Robarts Dakota R. , Paine-Cabrera Diego , Kotulkar Manasi , Venneman Kaitlyn K. , Gunewardena Sumedha , Corton J. Christopher , Lau Christopher , Foquet Lander , Bial Greg , Apte Udayan
Published: February 2023

Background

Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants with myriad adverse effects. While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are the most common contaminants, levels of replacement PFAS, such as perfluoro-2-methyl-3-oxahexanoic acid (GenX), are increasing. In rodents, PFOA, PFOS, and GenX have several adverse effects on the liver, including nonalcoholic fatty liver disease.

Objective

We aimed to determine human-relevant mechanisms of PFAS induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes.

Methods

Male humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. Liver and serum were collected for pathology and clinical chemistry, respectively. RNA-sequencing coupled with pathway analysis was used to determine molecular mechanisms.

Results

PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. PFOA had no significant changes in serum LDL/VLDL and total cholesterol. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed inhibition of NR1D1, a transcriptional repressor important in circadian rhythm, as the major common molecular change in all PFAS treatments. PFAS treated mice had significant nuclear localization of NR1D1.In silicomodeling showed PFOA, PFOS, and GenX potentially interact with the DNA-binding domain of NR1D1.

Discussion

These data implicate PFAS in circadian rhythm disruption via inhibition of NR1D1. These studies show that FRG humanized mice are a useful tool for studying the adverse outcome pathways of environmental pollutants on human hepatocytes in situ.
A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants
Authors: Patra Sourav Kumar , Carter Charles W.
Published: February 2023

Amino acyl tRNA synthetases or aaRSs play a key role in assuring the precision of protein translation. They are highly specific for their cognate amino acid and cognate tRNA substrates during protein synthesis, utilizing ATP to ensure that proper assignments are made between amino acid and anticodon. Specific aaRS for each amino acid are present in all cells. We describe a new zymography technique to qualitatively visualize and semi-quantitatively determine the amino acid activation capacity of each type of aaRS molecule by indirect colorimetric detection of released pyrophosphates during the formation of aminoacyl-AMP. Protein samples containing aaRS are subjected to Native PAGE, followed by incubation in buffer containing cognate amino acid and ATP for sufficient time to generate pyrophosphates (PPi) which are then converted to inorganic phosphates by pyrophosphatase treatment. Finally, the generated and localized phosphates around the aaRS protein inside the gel can be visualized after staining by ammonium molybdate and malachite green solution. This technique has been validated by inspecting the substrate specificities of specific aaRSs. This zymography technique is sufficiently sensitive to detect and authenticate activities of much (i.e., ~10-5-fold) less active aaRS “Urzymes”, to study alteration of activities of aaRS by various intrinsic or extrinsic factors and to screen aaRS-specific antimicrobial drugs.
SATB2 loss in inflammatory bowel disease-associated small intestinal metaplasia of the distal colon
Authors: Zeineldin Maged , Larman Tatianna C.
Published: February 2023

Epithelial metaplasia is a common adaptation to chronic inflammatory processes and can be associated with increased risk of dysplasia and cancer. The distal colon of patients with inflammatory bowel disease (IBD) commonly shows crypt architectural distortion and Paneth cell metaplasia (PCM), and IBD patients also carry increased risk of colitis-associated dysplasia and cancer (CAC). Loss of SATB2 expression (Special AT-rich binding 2 protein, a colon-restricted chromatin remodeler) has recently been shown to distinguish colitis-associated dysplasia and CAC from sporadic disease. Here we report non-diffuse heterogeneous patterns of SATB2 loss across non-dysplastic distal colon biopsies from IBD patients (n=20). This cohort was specifically curated to include biopsies with well-developed histologic features of villiform growth and PCM. Notably, CDX2 was strongly expressed and P53 showed a wild-type immunolabeling pattern across our non-dysplastic cohort, regardless of SATB2 immunolabeling pattern. Our findings fit with recent murine studies in which colon-specificSatb2deletion resulted in histologic conversion of colonic mucosa to small intestinal-like mucosa, including emergence of villi and Paneth cells. Taken together, we show that SATB2 loss is associated with a preneoplastic metaplastic response to chronic injury in human IBD and chronic colitis, reframing PCM more broadly as small intestinal metaplasia. We propose that inflammation-associated SATB2 loss mediates a remodeled chromatin landscape permissive for dysplasia and CAC.
Systematic Analysis of KRAS-ligand Interaction Modes and Flexibilities Reveals the Binding Characteristics
Authors: Zhao Zheng , Bohidar Niraja , Bourne Philip E.
Published: February 2023

KRAS, a common human oncogene, has been recognized as a critical drug target in treating multiple cancers. After four decades of effort, one allosteric KRAS drug (Sotorasib) has been approved, inspiring more KRAS-targeted drug research. Here we provide the features of KRAS binding pockets and ligand-binding characteristics of KRAS complexes using a structural systems pharmacology approach. Three distinct binding sites (conserved nucleotide-binding site, shallow Switch-I/II pocket, and allosteric Switch-II/α3 pocket) are characterized. Ligand-binding features are determined based on encoded KRAS-inhibitor interaction fingerprints. Finally, the flexibility of the three distinct binding sites to accommodate different potential ligands, based on MD simulation, is discussed. Collectively, these findings are intended to facilitate rational KRAS drug design.
Neuromuscular Basis ofDrosophilaLarval Rolling Escape Behavior
Authors: Cooney Patricia C. , Huang Yuhan , Li Wenze , Perera Dulanjana M. , Hormigo Richard , Tabachnik Tanya , Godage Isuru S. , Hillman Elizabeth M.C. , Grueber Wesley B. , Zarin Aref A.
Published: February 2023

When threatened by dangerous or harmful stimuli, animals engage in diverse forms of rapid escape behaviors. InDrosophilalarvae, one type of escape response involves C-shaped bending and lateral rolling followed by rapid forward crawling. The sensory circuitry that promotes larval escape has been extensively characterized; however, the motor programs underlying rolling are unknown. Here, we characterize the neuromuscular basis of rolling escape behavior. We used high-speed, volumetric, Swept Confocally-Aligned Planar Excitation (SCAPE) microscopy to image muscle activity during larval rolling. Unlike sequential peristaltic muscle contractions that progress from segment to segment during forward and backward crawling, the muscle activity progresses circumferentially during bending and rolling escape behavior. We propose that progression of muscular contraction around the larva’s circumference results in a transient misalignment between weight and the ground support forces, which generates a torque that induces stabilizing body rotation. Therefore, successive cycles of slight misalignment followed by reactive aligning rotation lead to continuous rolling motion. Supporting our biomechanical model, we found that disrupting the activity of muscle groups undergoing circumferential contraction progression lead to rolling defects. We use EM connectome data to identify premotor to motor connectivity patterns that could drive rolling behavior, and perform neural silencing approaches to demonstrate the crucial role of a group of glutamatergic premotor neurons in rolling. Our data reveal body-wide muscle activity patterns and putative premotor circuit organization for execution of the rolling escape response.

Significance Statement

To escape from dangerous stimuli, animals execute escape behaviors that are fundamentally different from normal locomotion. The rolling escape behavior of Drosophila larvae consists of C-shaped bending and rolling. However, the muscle contraction patterns that lead to rolling are poorly understood. We find that following the initial body bending, muscles contract in a circumferential wave around the larva as they enter the bend, maintaining unidirectional rolling that resembles cylinder rolling on a surface. We study the structure of motor circuits for rolling, inhibit different motor neurons to determine which muscles are essential for rolling, and propose circuit and biomechanical models for roll generation. Our findings provide insights into how motor circuits produce diverse motor behaviors.
Ozanimod Attenuates Human Cerebrovascular Endothelial Derived MMP-9 Activity and Preserves Barrier Properties Following In Vitro Acute Ischemic Injury
Authors: Wendt Trevor S. , Gonzales Rayna J.
Published: February 2023

Endothelial integrity is critical in mitigating a vicious cascade of secondary injuries following acute ischemic stroke (AIS). Matrix metalloproteinase-9 (MMP-9), a contributor to endothelial integrity loss, is elevated during stroke and is associated with worsened stroke outcome. We investigated the FDA approved selective sphingosine-1-phosphate receptor 1 (S1PR1) ligand, ozanimod, on the regulation/activity of MMP-9 as well as endothelial barrier components (PECAM-1, claudin-5, and ZO-1) in human brain microvascular endothelial cells (HBMECs) following hypoxia plus glucose deprivation (HGD). We previously reported that S1PR1 activation improves HBMEC integrity; however, specific mechanisms underlying S1PR1 involvement in barrier integrity have not been clearly elucidated. We hypothesized that ozanimod would attenuate an HGD-induced increase in MMP-9 activity which would concomitantly attenuate the loss of integral barrier components. Male HBMECs were treated with ozanimod (0.5nM) or vehicle and exposed to 3h normoxia (21% O2) or HGD (1% O2). Immunoblotting, zymography, qRT-PCR, and immunocytochemical labeling techniques assessed processes related to MMP-9 and barrier markers. We observed that HGD acutely increased MMP-9 activity and reduced claudin-5 and PECAM-1 levels, and ozanimod attenuated these responses. In situ analysis via PROSPER, suggested that attenuation of MMP-9 activity may be a primary factor in maintaining these integral barrier proteins. We also observed that HGD increased intracellular mechanisms associated with augmented MMP-9 activation, however ozanimod had no effect on these targeted factors. Thus, we conclude that ozanimod has the potential to attenuate HGD mediated decreases in HBMEC integrity in part by decreasing MMP-9 activity as well as preserving barrier properties.

Graphical Abstract
Microbiome induced complement synthesized in the gut protects against enteric infections
Authors: Wu Meng , Zheng Wen , Song Xinyang , Bao Bin , Wang Yuanyou , Ramanan Deepshika , Yang Daping , Liu Rui , Macbeth John C. , Do Elyza A. , Andrade Warrison A , Yang Tiandi , Cho Hyoung-Soo , Gazzaniga Francesca S. , Ilves Marit , Coronado Daniela , Thompson Charlotte , Hang Saiyu , Chiu Isaac M. , Moffitt Jeffrey R. , Hsiao Ansel , Mekalanos John J. , Benoist Christophe , Kasper Dennis L.
Published: February 2023

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection withCitrobacter rodentiumor enterohemorrhagicEscherichia coli,luminal C3 levels increase significantly and are required for protection.C. rodentiumis remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis ofC. rodentiumfunctions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals’ intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection.

Highlights

Gut complement component 3 (C3) is induced by the microbiome in healthy humans and mice at a microbiota-specific level.

Gut stromal cells located in intestinal lymphoid follicles are a major source of luminal C3

During enteric infections withCitrobacter rodentiumor enterohemorrhagicEscherichia coli,gut luminal C3 levels increase and are required for protection.

C. rodentiumis significantly more invasive of the gut epithelium in C3-deficient mice when compared to WT mice.

In the gut, C3-mediated opsonophagocytosis ofC. rodentiumfunctions to clear pathogens.
Membrane free-energy landscapes derived from atomistic dynamics explain nonuniversal cholesterol-induced stiffening
Authors: Fiorin Giacomo , Forrest Lucy R. , Faraldo-Gómez José D.
Published: February 2023

All lipid membranes have inherent morphological preferences and resist deformation. Yet adaptations in membrane shape can and do occur at multiple length scales. While this plasticity is crucial for cellular physiology, the factors controlling the morphological energetics of lipid bilayers and the dominant mechanisms of membrane remodeling remain unclear. An ongoing debate regarding the universality of the stiffening effect of cholesterol underscores the challenges facing this field, both experimentally and theoretically, even for simple lipid mixtures. On the computational side, we have argued that enhanced- sampling all-atom molecular dynamics simulations are uniquely suited for quantification of membrane conformational energetics, not only because they minimize a-priori assumptions, but also because they permit analysis of bilayers in deformed states. To showcase this approach, we examine reported inconsistencies between alternative experimental measurements of bending moduli for cholesterol-enriched membranes. Specifically, we analyze lipid bilayers with different chain saturation, and compute free-energy landscapes for curvature deformations distributed over areas from ∼5 to ∼60 nm2. These enhanced simulations, totaling over 100 microseconds of sampling time, enable us to directly quantify both bending and tilt moduli, and to dissect the contributing factors and molecular mechanisms of curvature generation at each length scale. Our results show that cholesterol effects are lipid-specific, in agreement with giantvesicle measurements, and explain why experiments probing nanometer scale lipid dynamics diverge. In summary, we demonstrate that quantitative structure-mechanics relationships can now be established for heterogenous membranes, paving the way for addressing open fundamental questions in cell membrane mechanics.

Significance

Elucidating the energetics and mechanisms of membrane remodeling is an essential step towards understanding cell physiology. This problem is challenging, however, because membrane bending involves both large-scale and atomic-level dynamics, which are difficult to measure simultaneously. A recent controversy regarding the stiffening effect of cholesterol, which is ubiquitous in animal cells, illustrates this challenge. We show how enhanced molecular-dynamics simulations can bridge this length-scale gap and reconcile seemingly incongruent observations. This approach facilitates a conceptual connection between lipid chemistry and membrane mechanics, thereby providing a solid basis for future research on remodeling phenomena, such as in membrane trafficking or viral infection.
Discovery of NRG1-VII: a novel myeloid-derived class of NRG1 isoforms
Authors: Berrocal-Rubio Miguel Ángel , Pawer Yair David Joseph , Dinevska Marija , De Paoli-Iseppi Ricardo , Widodo Samuel S. , Gleeson Josie , Rajab Nadia , De Nardo Will , Hallab Jeannette , Li Anran , Mantamadiotis Theo , Clark Michael B. , Wells Christine A.
Published: February 2023

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut, brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1, designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another, through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and iPSC-derived macrophages. Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage, including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1, suggesting that NRG1-VII is the most common class of NRG1 in most adult human tissues, except brain. The size and structure of type VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However, the specific roles of type VII variants in tissue homeostasis and repair have not yet been determined.
An Innovative Non-Hormonal Strategy Targeting Redox Active Metals to Down-Regulate Estrogen-, Progesterone-, Androgen- and Prolactin-Receptors in Breast Cancer
Authors: Shehadeh-Tout Faten , Milioli Heloisa H. , Roslan Suraya , Jansson Patric J. , Dharmasivam Mahendiran , Graham Dinny , Anderson Robin , Wijesinghe Tharushi , Azad Mahan Gholam , Richardson Des R. , Kovacevic Zaklina
Published: February 2023

Estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) targeted by tamoxifen. However, tamoxifen resistance is a major problem. An important mechanism of resistance is the activation of EGFR/HER2/HER3 signaling and other hormone receptors (androgen receptor (AR), progesterone receptor (PR), prolactin receptor (PRL-R)) that intrinsically activate ER-α. Hence, therapeutics targeting multiple receptors, rather than ER-α alone, would be extremely useful and may overcome tamoxifen resistance. This study examined the activity of redox-active di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of crucial hormone receptors, their co-factors, and key resistance pathways in ER-α-positive BC. Strikingly, DpC differentially regulated 106 estrogen-response genes with Sankey diagram analysis demonstrating this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namelyER,PR,AR, andPRL-R. Mechanistic dissection demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. Ablation of the metal-binding site in the thiosemicarbazone totally prevented its suppressive activity, demonstrating a unique non-hormonal mechanism. DpC and Dp44mT also inhibited EGFR, HER2, and HER3 activation, their downstream signaling, and the expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1.In vivo,DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. In conclusion, through a bespoke non-hormonal mechanism targeting redox active metals, Dp44mT and DpC disrupt multiple key inter-receptor interactions between PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.
Oral administration of 4-methylumbelliferone reduces glial scar and promotes anatomical plasticity
Authors: Štepánková Kateřina , Chudíčková Milada , Šimková Zuzana , Martinez-Varea Noelia , Kubinová Šárka , Urdzíková Lucia Machová , Jendelová Pavla , Kwok Jessica C F
Published: February 2023

Following a spinal cord injury (SCI), chondroitin sulfate proteoglycans (CSPGs) are up-regulated at the glial scar inhibiting neuroregeneration. Under normal physiological condition, CSPGs interact with hyaluronan (HA) and other extracellular matrix on neuronal surface forming a macromolecular structure called perineuronal nets (PNNs) which regulate neuroplasticity. 4-methylumbelliferone (4-MU) has been used previously to down-regulate HA synthesis but not been tested in SCI. In this study, we have evaluated the effect of 4-MU, an inhibitor of HA, in a chronic contusion model of SCI in rats. At a dose of 1.2 g/kg/day of 4-MU, we observed not only the reduction of HA in the uninjured spinal cords after 60 days of 4-MU administration, but also a down-regulation of CS glycosaminoglycans (CS-GAGs). In order to assess the effect of 4-MU in chronic SCI, rats with T8 spinal contusion injury were fed with 4-MU or placebo for 8 weeks in combination with daily treadmill rehabilitation for 16 weeks to promote neuroplasticity. 4-MU treatment promoted significant sprouting of 5-hydroxytryptamine (5-HT) positive fibres into ventral horns and reduced the HA synthesis by astrocytes around the lesion site. While 4-MU reduced astrogliosis in chronic stage of SCI, the current dose was not sufficient to down-regulate the increased production of CS-GAGs or behavioural performance. Together, these data suggest that oral treatment with 4-MU is able to induce anatomical plasticity but further adjustment on the dosage will be required to benefit functional recovery after SCI.
Conformational plasticity and allosteric communication networks govern Shelterin protein TPP1 binding to human telomerase
Authors: Aureli Simone , Raniolo Stefano , Limongelli Vittorio
Published: February 2023

The molecular binding interaction between the Shelterin complex protein TPP1 and human telomerase enzyme (TERT) triggers the telomerase maintenance mechanism that marks cell lifespan. The TPP1’s structural element deputed to bind TERT is the OB-domain, which is able to interact with TERT’s hTEN (TPP1 binding telomerase domain) through the TEL-patch, a group of amino acids whose mutations provoke harsh pathologies. Indeed, aberrations in the formation of TPP1-TERT het-erodimer can lead to severe diseases like Hoyeraal-Hreidarsson syndrome (HHS), whose patients are affected by short telomeres and extremely poor life expectancy. In the present study, we provide a thorough characterization of the structural properties of the TPP1’s OB-domain by combining data coming from microsecond-long molecular dynamics calculations, time-series analyses, and graph-based networks. Our results show that the conformational plasticity of the TPP1’s TEL-patch region is influenced by a network of long-range amino acid communications, needed for the proper TPP1-hTEN binding. Furthermore, we reveal that in the Glu169Δand Lys170ΔTPP1 variants, responsible for HHS, the plasticity of the TEL-patch region is reduced, affecting the correct binding to hTEN and in turn the telomere processivity, which eventually leads to accelerated ageing of affected cells. Our study provides an unprecedented structural basis for the design of TPP1-targeting ligands with therapeutic potential against cancer and telomerase deficiency diseases.
PermaNet® Dual, a new deltamethrin-chlorfenapyr mixture net, shows improved efficacy against pyrethroid-resistantAnopheles gambiae sensu latoin southern Benin
Authors: Syme Thomas , N’dombidjé Boris , Gbegbo Martial , Todjinou Damien , Ariori Victoria , De Vos Patricia , Pigeon Olivier , Ngufor Corine
Published: February 2023

Pyrethroid-chlorfenapyr nets have demonstrated improved entomological and epidemiological impact in trials across Africa. This is driving increased demand for this novel net class in malaria endemic countries. PermaNet® Dual is a new deltamethrin-chlorfenapyr net developed by Vestergaard Sàrl to provide more options to malaria control programmes. We performed an experimental hut trial to evaluate the efficacy of PermaNet® Dual against wild, free-flying pyrethroid-resistantAnopheles gambiae sensu latoin Covè, Benin. PermaNet® Dual induced superior levels of mosquito mortality compared to a pyrethroid-only net and a pyrethroid-piperonyl butoxide net both when unwashed (77% with PermaNet® Dual vs. 23% with PermaNet® 2.0 and 56% with PermaNet® 3.0, p<0.001) and after 20 standardised washes (75% with PermaNet® Dual vs. 14% with PermaNet® 2.0 and 30% with PermaNet® 3.0, p<0.001). Using a provisional non-inferiority margin defined by the World Health Organisation, PermaNet® Dual was also non-inferior to a pyrethroid-chlorfenapyr net that has demonstrated improved public health value (Interceptor® G2), for vector mortality (79% vs. 76%, OR=0.854, 95% CIs: 0.703–1.038) but not for blood-feeding protection (35% vs. 26%, OR=1.445, 95% CIs: 1.203–1.735). PermaNet® Dual presents an additional option of this highly effective net class for improved control of malaria transmitted by pyrethroid-resistant mosquitoes.
Social affective behaviors among female rats involve the basolateral amygdala and insular cortex
Authors: Djerdjaj Anthony , Rieger Nathaniel S. , Brady Bridget H. , Carey Bridget N. , Ng. Alexandra J. , Christianson John P.
Published: February 2023

The ability to detect, appraise, and respond to another’s emotional state is essential to social affective behavior. This is mediated by a network of brain regions responsible for integrating external cues with internal states to orchestrate situationally appropriate behavioral responses. The basolateral amygdala (BLA) and the insular cortex are reciprocally connected regions involved in social cognition and prior work in male rats revealed their contributions to social affective behavior. We investigated the functional role of these regions in female rats in a social affective preference (SAP) test in which experimental rats approach stressed juvenile but avoid stressed adult conspecifics. In separate experiments, the BLA or the insula were inhibited by local infusion of muscimol (100ng/side in 0.5μL saline) or vehicle prior to SAP tests. In both regions, muscimol interfered with preference for the stressed juvenile and naive adult, indicating that these regions are necessary for appropriate social affective behavior. In male rats, SAP behavior requires insular oxytocin but there are noteworthy sex differences in the oxytocin receptor distribution in rats. Oxytocin (500nM) administered to the insula did not alter social behavior but oxytocin infusions to the BLA increased social interaction. In sum, female rats appear to use the same BLA and insula regions for social affective behavior but sex differences exist in contribution of oxytocin in the insula.
An unbiased, automated platform for scoring dopaminergic neurodegeneration inC. elegans
Authors: Clark Andrew S. , Kalmanson Zachary , Morton Katherine , Hartman Jessica , Meyer Joel , San-Miguel Adriana
Published: February 2023

Caenorhabditis elegans(C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration inC. elegans. The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in theC. eleganshead and the pixel size of the user’s camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 19 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.
Effects of pathogen sexual reproduction on the evolutionary and epidemiological control provided by deployment strategies for two major resistance genes in agricultural landscapes
Authors: Zaffaroni Marta , Rimbaud Loup , Rey Jean-François , Papaïx Julien , Fabre Frédéric
Published: February 2023

Resistant cultivars are of value for protecting crops from disease, but can be rapidly overcome by pathogens. Several strategies have been proposed to delay pathogen adaptation (evolutionary control), while maintaining effective protection (epidemiological control). Resistance genes can bei) combined in the same cultivar (pyramiding),ii) deployed in different cultivars sown in the same field (mixtures) or in different fields (mosaics), oriii) alternated over time (rotations). The outcomes of these strategies have been investigated principally in pathogens displaying pure clonal reproduction, but sexual reproduction may promote the emergence of superpathogens adapted to all the resistance genes deployed.

We improved the spatially explicit stochastic modellandsepito include pathogen sexual reproduction, and then investigate the effect of sexual reproduction on evolutionary and epidemiological outcomes across deployment strategies for two major resistance genes.

Sexual reproduction only favours the establishment of a superpathogen when single mutant pathogens are present together at a sufficiently high frequency, as in mosaic and mixture strategies.

We concluded that, although sexual reproduction may promote the establishment of a superpathogen, it did not affect the optimal strategy recommendations for a wide range of mutation probabilities, associated fitness costs, and landscape organisations (notably the cropping ratio of resistant fields).
Thermal conditioning of quail embryos has transgenerational and reversible long-term effects
Authors: Carvalho Anaïs Vitorino , Hennequet-Antier Christelle , Rouger Romuald , Delaveau Joël , Bordeau Thierry , Crochet Sabine , Couroussé Nathalie , Pitel Frederique , Collin Anne , Coustham Vincent
Published: February 2023

In the current context of global warming, thermal manipulation of avian embryos has received increasing attention as a strategy to promote heat tolerance in avian species by simply increasing the egg incubation temperature. However, because of their likely epigenetic origin, thermal manipulation effects may last more than one generation with consequences for the poultry industry. We found that thermal manipulation repeated during 4 generations had an effect on hatchability, body weight, and weight of eggs laid in Japanese quails, with some effects increasing in importance over generations. Moreover, we show that the effects on body weight and egg weight can be transmitted transgenerationally, suggesting non-genetic inheritance mechanisms. This hypothesis is reinforced by the observed reversion of the effect on growth after five unexposed generations. Interestingly, we demonstrate a non-transgenerational effect of thermal manipulation on heat tolerance few days after hatch. In conclusion, our multigenerational study suggests a quantitative, transgenerational and reversible effect of thermal manipulation.
Development of attention-based robust deep learning model for tongue diagnosis by smartphone
Authors: Ma Changzheng , Gao Chaofei , Hou Siyu , Li Shao
Published: February 2023

Compared with tongue diagnosis using tongue image analyzers, tongue diagnosis by smartphone has great advantages in convenience and cost for universal health monitoring, but its accuracy is affected by the shooting conditions of smartphones. Developing deep learning models with high accuracy and robustness to changes in the shooting environment for tongue diagnosis by smartphone and determining the influence of environmental changes on accuracy are necessary. In our study, a dataset of 9003 images was constructed after image pre-processing and labeling. Next, we developed an attention-based deep learning model (Deep Tongue) for 8 subtasks of tongue diagnosis, including the spotted tongue, teeth-marked tongue, and fissure tongue et al, which the average AUC of was 0.90, higher than the baseline model (ResNet50) by 0.10. Finally, we analyzed the objective reasons, the brightness of the environment and the hue of images, affecting the accuracy of tongue diagnosis by smartphone through a consistency experiment of direct subject inspection and tongue image inspection. Finally, we determined the influence of environmental changes on accuracy to quantify the robustness of the Deep Tongue model through simulation experiments. Overall, the Deep Tongue model achieved a higher and more stable classification accuracy of seven tongue diagnosis tasks in the complex shooting environment of the smartphone, and the classification of tongue coating (yellow/white) was found to be sensitive to the hue of the images and therefore unreliable without stricter shooting conditions and color correction.