Epigenetic weapons in plant-herbivore interactions: Sulforaphane disrupts histone deacetylases, gene expression, and larval development in Spodoptera exigua while the specialist feeder Trichoplusia ni is largely resistant to these effects.

Cruciferous plants produce sulforaphane (SFN), an inhibitor of nuclear histone deacetylases (HDACs). In humans and other mammals, the consumption of SFN alters enzyme activities, DNA-histone binding, and gene expression within minutes. However, the ability of SFN to act as an HDAC inhibitor in nature, disrupting the epigenetic machinery of insects feeding on these plants, has not been explored. Here, we demonstrate that SFN consumed in the diet inhibits the activity of HDAC enzymes and slows the development of the generalist grazer Spodoptera exigua, in a dose-dependent fashion. After consuming SFN for seven days, the activities of HDAC enzymes in S. exigua were reduced by 50%. Similarly, larval mass was reduced by 50% and pupation was delayed by 2-5 days, with no additional mortality. Similar results were obtained when SFN was applied topically to eggs. RNA-seq analyses confirm that SFN altered the expression of thousands of genes in S. exigua. Genes associated with energy conversion pathways were significantly downregulated while those encoding for ribosomal proteins were dramatically upregulated in response to the consumption of SFN. In contrast, the co-evolved specialist feeder Trichoplusia ni was not negatively impacted by SFN, whether it was consumed in their diet at natural concentrations or applied topically to eggs. The activities of HDAC enzymes were not inhibited and development was not disrupted. In fact, SFN exposure sometimes accelerated T. ni development. RNA-seq analyses revealed that the consumption of SFN alters gene expression in T. ni in similar ways, but to a lesser degree, compared to S. exigua. This apparent resistance of T. ni can be overwhelmed by unnaturally high levels of SFN or by exposure to more powerful pharmaceutical HDAC inhibitors. These results demonstrate that dietary SFN interferes with the epigenetic machinery of insects, supporting the hypothesis that plant-derived HDAC inhibitors serve as "epigenetic weapons" against herbivores.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 Å resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

Radial glia promote microglial development through integrin αVß8 -TGFß1 signaling.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFß1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFß1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFß signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

Photoinduced bidirectional switching in lipid membranes containing azobenzene glycolipids.

Following the reaction of biological membranes to external stimuli reveals fundamental insights into cellular function. Here, self-assembled lipid monolayers act as model membranes containing photoswitchable azobenzene glycolipids for investigating structural response during isomerization by combining Langmuir isotherms with X-ray scattering. Controlled in-situ trans/cis photoswitching of the azobenzene N = N double bond alters the DPPC monolayer structure, causing reproducible changes in surface pressure and layer thickness, indicating monolayer reorientation. Interestingly, for monolayers containing azobenzene glycolipids, along with the expected DPPC phase transitions an additional discontinuity is observed. The associated reorintation represents a crossover point, with the surface pressure and layer thickness changing in opposite directions above and below. This is evidence that the azobenzene glycolipids themselves change orientation within the monolayer. Such behaviour suggests that azobenzene glycolipids can act as a bidirectional switch in DPPC monolayers providing a tool to investigate membrane structure-function relationships in depth.

Loss of control eating in relation to blood pressure among adolescent girls with elevated anxiety at-risk for excess weight gain.

Loss of control (LOC)-eating, excess weight, and anxiety are robustly linked, and are independently associated with markers of poorer cardiometabolic health, including hypertension. However, no study has examined whether frequency of LOC-eating episodes among youth with anxiety symptoms and elevated weight status may confer increased risk for hypertension. We examined the relationship between LOC-eating frequency and blood pressure among 39 adolescent girls (14.9 ± 1.8 years; body mass index [BMI] = 29.9 ± 5.6; 61.5 % White; 20.5 % African American/Black; 5 % Multiple Races; 2.5 % Asian; 12.8 % Hispanic/Latino; 30.8 % with reported LOC-eating) with elevated anxiety and above average BMI who enrolled in a clinical trial aimed at preventing excess weight gain. LOC-eating over the past three months was assessed via clinical interview, and blood pressure (systolic and diastolic) was measured with an automatic blood pressure monitor. Adjusting for age, fat mass, and height, LOC-eating episode frequency was significantly, positively associated with diastolic blood pressure (ß = 0.38, p = 0.02), but not with systolic blood pressure (ß = 0.13, p = 0.41). Replication studies, with larger sample sizes, participants of varying weight-strata, and prospective data are required to elucidate the relationship between LOC-eating and cardiovascular functioning in youth with elevated anxiety.

Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models.

The recent proliferation of new Cre and CreER recombinase lines provides researchers with a diverse toolkit to study microglial gene function. To determine how best to apply these lines in studies of microglial gene function, a thorough and detailed comparison of their properties is needed. Here, we examined four different microglial CreER lines (Cx3cr1CreER(Litt), Cx3cr1CreER(Jung), P2ry12CreER, Tmem119CreER), focusing on (1) recombination specificity; (2) leakiness - degree of non-tamoxifen recombination in microglia and other cells; (3) efficiency of tamoxifen-induced recombination; (4) extra-neural recombination -the degree of recombination in cells outside the CNS, particularly myelo/monocyte lineages (5) off-target effects in the context of neonatal brain development. We identify important caveats and strengths for these lines which will provide broad significance for researchers interested in performing conditional gene deletion in microglia. We also provide data emphasizing the potential of these lines for injury models that result in the recruitment of splenic immune cells.

Trends in Drug Tests among Children: A 22-Year Retrospective Analysis.

There are several pathophysiological outcomes associated with substance abuse including metabolic disbalance, neurodegeneration, and disordered redox. Drug use in pregnant women is a topic of great concern due to developmental harm which may occur during gestation and the associated complications in the neonate after delivery. We sought to determine what the trajectory of drug use is like in children aged 0-4 years and mothers of neonates. Urine drug screen (UDS) results were obtained of our target demographic during 1998-2011 and 2012-2019 from LSU Health Sciences Center in Shreveport (LSUHSC-S). Statistical analysis was performed using R software. We observed an increase in cannabinoid-positive UDS results in both Caucasian (CC) and African American (AA) groups between 1998-2011 and 2012-2019 periods. Cocaine-positive UDS results decreased in both cohorts. CC children had higher UDS positive results for opiates, benzodiazepines, and amphetamines, while AA children had a higher percentage for illicit drugs such as cannabinoids and cocaine. Neonate's mothers had similar UDS trends to that in children during 2012-2019. Overall, while percentage of positive UDS results for both AA and CC 0-4 year old children started to decline for opiate, benzodiazepine, and cocaine during 2012-2019, cannabinoid- and amphetamine (CC)-positive UDS steadily increased. These results suggest a shift in the type of drug use by mothers from opiates, benzodiazepines, and cocaine to cannabinoids and/or amphetamines. We also observed that 18-year-old females who tested positive for opiates, benzodiazepine, or cocaine had higher than average chances of testing positive for cannabinoids later in life.

Learning metric volume estimation of fruits and vegetables from short monocular video sequences.

We present a novel approach for extracting metric volume information of fruits and vegetables from short monocular video sequences and associated inertial data recorded with a hand-held smartphone. Estimated segmentation masks from a pre-trained object detector are fused with the predicted change in relative pose obtained from the inertial data to predict the class and volume of the objects of interest. Our approach works with simple RGB video frames and inertial data which are readily available from modern smartphones. It does not require reference objects of known size in the video frames. Using a balanced validation dataset, we achieve a classification accuracy of 95% and a mean absolute percentage error for the volume prediction of 16% on untrained objects, which is comparable to state-of-the-art results requiring more elaborated data recording setups. A very accurate estimation of the model uncertainty is achieved through ensembling and the use of Gaussian negative log-likelihood loss. The dataset used in our experiments including ground-truth volume information is available at https://sst.aau.at/cns/datasets.

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvß5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

Effect of Anionic Lipids on Mammalian Plasma Cell Membrane Properties.

The effect of lipid composition on models of the inner leaflet of mammalian cell membranes has been investigated. Grazing incidence X-ray diffraction and X-ray and neutron reflectivity have been used to characterize lipid packing and solvation, while electrochemical and infrared spectroscopic methods have been employed to probe phase behavior in an applied electric field. Introducing a small quantity of the anionic lipid dimyristoylphosphatidylserine (DMPS) into bilayers of zwitterionic dimyristoylphosphatidylethanolamine (DMPE) results in a significant change in the bilayer response to an applied field: the tilt of the hydrocarbon chains increases before returning to the original tilt angle on detachment of the bilayer. Equimolar mixtures, with slightly closer chain packing, exhibit a similar but weaker response. The latter also tend to incorporate more solvent during this electrochemical phase transition, at levels similar to those of pure DMPS. Reflectivity measurements reveal greater solvation of lipid layers for DMPS > 30 mol %, matching the greater propensity for DMPS-rich bilayers to incorporate water. Taken together, the data indicate that the range of 10-35 mol % DMPS provides optimum bilayer properties (in flexibility and function as a barrier), which may explain why the DMPS content of cell membranes tends to be found within this range.

Advice on describing Bayesian analysis of neutron and X-ray reflectometry.

As a result of the availability of modern software and hardware, Bayesian analysis is becoming more popular in neutron and X-ray reflectometry analysis. The understandability and replicability of these analyses may be harmed by inconsistencies in how the probability distributions central to Bayesian methods are represented in the literature. Herein advice is provided on how to report the results of Bayesian analysis as applied to neutron and X-ray reflectometry. This includes the clear reporting of initial starting conditions, the prior probabilities, the results of any analysis and the posterior probabilities that are the Bayesian equivalent of the error bar, to enable replicability and improve understanding. It is believed that this advice, grounded in the authors' experience working in the field, will enable greater analytical reproducibility in the work of the reflectometry community, and improve the quality and usability of results.

Influence of the Lipid Backbone on Electrochemical Phase Behavior.

Sphingolipids are an important class of lipids found in mammalian cell membranes with important structural and signaling roles. They differ from another major group of lipids, the glycerophospholipids, in the connection of their hydrocarbon chains to their headgroups. In this study, a combination of electrochemical and structural methods has been used to elucidate the effect of this difference on sphingolipid behavior in an applied electric field. N-Palmitoyl sphingomyelin forms bilayers of similar coverage and thickness to its close analogue di-palmitoyl phosphatidylcholine. Grazing incidence diffraction data show slightly closer packing and a smaller chain tilt angle from the surface normal. Electrochemical IR results at low charge density show that the difference in tilt angle is retained on deposition to form bilayers. The bilayers respond differently to increasing electric field strength: chain tilt angles increase for both molecules, but sphingomyelin chains remain tilted as field strength is further increased. This behavior is correlated with disruption of the hydrogen-bonding network of small groups of sphingomyelin molecules, which may have significance for the behavior of molecules in lipid rafts in the presence of strong fields induced by ion gradients or asymmetric distribution of charged lipids.

Addressing Anxiety and Stress for Healthier Eating in Teens (ASSET): A Pilot Randomized Controlled Trial Protocol for Reducing Anxiety, Disinhibited Eating, Excess Weight Gain, and Cardiometabolic Risk in Adolescent Girls.

(1) Background: Standard-of-care lifestyle interventions show insufficient effectiveness for the prevention and treatment of excess weight and its associated cardiometabolic health concerns in adolescents, necessitating more targeted preventative approaches. Anxiety symptoms are common among adolescents, especially girls at risk for excess weight gain, and have been implicated in the onset and maintenance of disinhibited eating. Thus, decreasing elevated anxiety in this subset of adolescent girls may offer a targeted approach to mitigating disinhibited eating and excess weight gain to prevent future cardiometabolic health problems. (2) Methods: The current paper describes the protocol for a multisite pilot and feasibility randomized controlled trial of group cognitive behavioral therapy (CBT) and group interpersonal psychotherapy (IPT) in N = 40 adolescent girls (age 12-17 years) with elevated anxiety symptoms and body mass index (BMI; kg/m2) ≥ 75th percentile for age/sex. (3) Results: Primary outcomes are multisite feasibility of recruitment, protocol procedures, and data collection, intervention fidelity, retention at follow-ups, and acceptability of interventions and study participation. (4) Conclusions: Findings will inform the protocol for a future fully-powered multisite randomized controlled trial to compare CBT and IPT efficacy for reducing excess weight gain and preventing adverse cardiometabolic trajectories, as well as to evaluate theoretically-informed treatment moderators and mediators.

Structural investigation of sulfobetaines and phospholipid monolayers at the air-water interface.

Mixtures of sulfobetaine based lipids with phosphocholine phospholipids are of interest in order to study the interactions between zwitterionic surfactants and the phospholipids present in cell membranes. In this study we have investigated the structure of mixed monolayers of sulfobetaines and phosphocholine phospholipids. The sulfobetaine used has a single 18-carbon tail, and is referred to as SB3-18, and the phospholipid used is DMPC. Surface pressure-area isotherms of the samples were used to determine whether any phase transitions were present during the compression of the monolayers. Neutron and X-ray reflectometry were then used to investigate the structure of these monolayers perpendicular to the interface. We found that the average headgroup and tail layer thickness was reasonably consistent across all mixtures, with a variation of less than 3 Å reported in the total thickness of the monolayers at each surface pressure. However, by selective deuteration of the two components of the monolayers, it was found that the two components have different tail layer thicknesses. For the mixture with equal compositions of DMPC and SB3-18 or with a higher composition of DMPC the tail tilts were found to be constant, resulting in a greater tail layer thickness for SB3-18 due to its longer tail. For the mixture higher in SB3-18 this was not the case, the tail tilt angle for the two components was found to be different and DMPC was found to have a greater tail layer thickness than SB3-18 as a result.

Low-field MRI: Clinical promise and challenges.

Modern MRI scanners have trended toward higher field strengths to maximize signal and resolution while minimizing scan time. However, high-field devices remain expensive to install and operate, making them scarce outside of high-income countries and major population centers. Low-field strength scanners have drawn renewed academic, industry, and philanthropic interest due to advantages that could dramatically increase imaging access, including lower cost and portability. Nevertheless, low-field MRI still faces inherent limitations in image quality that come with decreased signal. In this article, we review advantages and disadvantages of low-field MRI scanners, describe hardware and software innovations that accentuate advantages and mitigate disadvantages, and consider clinical applications for a new generation of low-field devices. In our review, we explore how these devices are being or could be used for high acuity brain imaging, outpatient neuroimaging, MRI-guided procedures, pediatric imaging, and musculoskeletal imaging. Challenges for their successful clinical translation include selecting and validating appropriate use cases, integrating with standards of care in high resource settings, expanding options with actionable information in low resource settings, and facilitating health care providers and clinical practice in new ways. By embracing both the promise and challenges of low-field MRI, clinicians and researchers have an opportunity to transform medical care for patients around the world. LEVEL OF EVIDENCE: 5 TECHNICAL EFFICACY: Stage 6.

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.