PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

proGenomes3: approaching one million accurately and consistently annotated high-quality prokaryotic genomes.

The interpretation of genomic, transcriptomic and other microbial 'omics data is highly dependent on the availability of well-annotated genomes. As the number of publicly available microbial genomes continues to increase exponentially, the need for quality control and consistent annotation is becoming critical. We present proGenomes3, a database of 907 388 high-quality genomes containing 4 billion genes that passed stringent criteria and have been consistently annotated using multiple functional and taxonomic databases including mobile genetic elements and biosynthetic gene clusters. proGenomes3 encompasses 41 171 species-level clusters, defined based on universal single copy marker genes, for which pan-genomes and contextual habitat annotations are provided. The database is available at http://progenomes.embl.de/.

A strategic approach for efficient cryo-EM grid optimization using design of experiments.

In recent years, cryo-electron microscopy (cryo-EM) has become a practical and effective method of determining structures at previously unattainable resolutions due to advances in detection, automation, and data processing. However, sample preparation remains a major bottleneck in the cryo-EM workflow. Even after the arduous process of biochemical sample optimization, it often takes several iterations of grid vitrification and screening to determine the optimal grid freezing parameters that yield suitable ice thickness and particle distribution for data collection. Since a high-quality sample is imperative for high-resolution structure determination, grid optimization is a vital step. For researchers who rely on cryo-EM facilities for grid screening, each iteration of this optimization process may delay research progress by a matter of months. Therefore, a more strategic and efficient approach should be taken to ensure that the grid optimization process can be completed in as few iterations as possible. Here, we present an implementation of Design of Experiments (DOE) to expedite and strategize the grid optimization process. A Fractional Factorial Design (FFD) guides the determination of a limited set of experimental conditions which can model the full parameter space of interest. Grids are frozen with these conditions and screened for particle distribution and ice thickness. Quantitative scores are assigned to each of these grid characteristics based on a qualitative rubric. Input conditions and response scores are used to generate a least-squares regression model of the parameter space in JMP, which is used to determine the conditions which should, in theory, yield optimal grids. Upon testing this approach on apoferritin and L-glutamate dehydrogenase on both the Vitrobot Mark IV and the Leica GP2 plunge freezers, the resulting grid conditions reliably yielded grids with high-quality ice and particle distribution that were suitable for collecting large overnight datasets on a Krios. We conclude that a DOE-based approach is a cost-effective and time-saving tool for cryo-EM grid preparation.

Double-Balloon Catheter-Mediated Transarterial Chemotherapy Delivery in a Swine Model: A Mechanism Recruiting the Vasa Vasorum for Localized Therapies.

PURPOSE: Treatment of hypovascular tumors, such as pancreatic adenocarcinoma, is challenging owing to inefficient drug delivery. This report examines the potential mechanism of localized drug delivery via transarterial microperfusion (TAMP) using a proprietary adjustable double-balloon occlusion catheter in a porcine model. MATERIALS AND METHODS: Adult Yorkshire swine (N = 21) were used in the Institutional Animal Care & Use Committee-approved protocols. The RC-120 catheter (RenovoRx, Los Altos, California) was positioned into visceral, femoral, and pulmonary arteries with infusion of methylene blue dye, gemcitabine, or gold nanoparticles. Transmural delivery was compared under double-balloon occlusion with and without side-branch exclusion, single-balloon occlusion, and intravenous delivery. Intra-arterial pressure and vascular histologic changes were assessed. RESULTS: Infusion with double-balloon occlusion and side-branch exclusion provided increased intra-arterial pressure in the isolated segment and enhanced perivascular infusate penetration with minimal vascular injury. Infusates were predominantly found in the vasa vasorum by electron microscopy. CONCLUSIONS: TAMP enhanced transmural passage mediated by localized increase in arterial pressure via vasa vasorum.

The P5-type ATPase ATP13A1 modulates major histocompatibility complex I-related protein 1 (MR1)-mediated antigen presentation.

The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included ß2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.

Metronidazole for Prevention of Pelvic Cellulitis and Abscess after Laparoscopic Hysterectomy: A Triple-blinded, Randomized, Placebo-controlled Clinical Trial.

STUDY OBJECTIVE: To determine whether a postoperative 5-day treatment schedule with vaginal metronidazole added to conventional antibiotic prophylaxis with 2 g cefazolin modifies the risk of pelvic cellulitis (PC) and pelvic abscess (PA) after total laparoscopic hysterectomy (TLH). DESIGN: A randomized, controlled, triple-blind, multicenter clinical trial. SETTING: Two centers dedicated to minimally invasive gynecologic surgery in Colombia. PATIENTS: A total of 574 patients were taken to TLH because of benign diseases. INTERVENTION: Patients taken to TLH were divided into 2 groups (treatment group, cefazolin 2 g intravenous single dose before surgery + metronidazole vaginal ovules for 5 days postoperatively, control group: cefazolin 2 g intravenous single dose + placebo vaginal ovules for 5 days postoperatively). MEASUREMENTS AND MAIN RESULTS: The absolute frequency (AF) of PC and PA and their relationship with the presence of bacterial vaginosis (BV) were measured. There was no difference in AF of PC (AF, 2/285 [0.7%] vs 5/284 [1.7%] in the treatment and placebo groups, respectively; risk ratio, 1.75; 95% confidence interval, 0.54-5.65; p = .261), nor for PA (AF, 0/285 [0%] vs 2/289 [0.7%]; p = .159, in the treatment and placebo groups, respectively). The incidence of BV was higher in the metronidazole group than the placebo group (42.5% vs 33.4%, p = .026). CONCLUSION: The use of vaginal metronidazole ovules during the first 5 days in postoperative TLH added to conventional cefazolin prophylaxis does not prevent the development of PC or PA, regardless of the patient's diagnosis of BV.

Ligand-dependent downregulation of MR1 cell surface expression.

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.

The Role of Citicoline and Coenzyme Q10 in Retinal Pathology.

Ocular neurodegenerative diseases such as glaucoma, diabetic retinopathy, and age-related macular degeneration are common retinal diseases responsible for most of the blindness causes in the working-age and elderly populations in developed countries. Many of the current treatments used in these pathologies fail to stop or slow the progression of the disease. Therefore, other types of treatments with neuroprotective characteristics may be necessary to allow a more satisfactory management of the disease. Citicoline and coenzyme Q10 are molecules that have neuroprotective, antioxidant, and anti-inflammatory properties, and their use could have a beneficial effect in ocular neurodegenerative pathologies. This review provides a compilation, mainly from the last 10 years, of the main studies that have been published on the use of these drugs in these neurodegenerative diseases of the retina, analyzing the usefulness of these drugs in these pathologies.

INHBA is a mediator of aggressive tumor behavior in HER2+ basal breast cancer.

BACKGROUND: Resistance to HER2-targeted therapeutics remains a significant clinical problem in HER2+ breast cancer patients with advanced disease. This may be particularly true for HER2+ patients with basal subtype disease, as recent evidence suggests they receive limited benefit from standard of care HER2-targeted therapies. Identification of drivers of resistance and aggressive disease that can be targeted clinically has the potential to impact patient outcomes. METHODS: We performed siRNA knockdown screens of genes differentially expressed between lapatinib-responsive and -resistant HER2+ breast cancer cells, which corresponded largely to luminal versus basal subtypes. We then validated hits in 2-d and 3-d cell culture systems. RESULTS: Knockdown of one of the genes, INHBA, significantly slowed growth and increased sensitivity to lapatinib in multiple basal HER2+ cell lines in both 2-d and 3-d cultures, but had no effect in luminal HER2+ cells. Loss of INHBA altered metabolism, eliciting a shift from glycolytic to oxidative phosphorylative metabolism, which was also associated with a decrease in tumor invasiveness. Analysis of breast cancer datasets showed that patients with HER2+ breast cancer and high levels of INHBA expression had worse outcomes than patients with low levels of INHBA expression. CONCLUSIONS: Our data suggest that INHBA is associated with aggressiveness of the basal subtype of HER2+ tumors, resulting in poor response to HER2-targeted therapy and an invasive phenotype. We hypothesize that targeting this pathway could be an effective therapeutic strategy to reduce invasiveness of tumor cells and to improve therapeutic response.

Sex-specific role of the optic gland in octopus maya: A transcriptomic analysis.

The optic glands (OG) of cephalopods are a source of molecules associated with the control of reproductive traits and lifecycle events such as sexual maturation, reproductive behavior, feeding, parental care, and senescence. However, little is known about the role of the optic gland in Octopus maya adults during mating and egg laying. RNA sequencing, de novo transcriptome assembly, ubiquity and differential expression analysis were performed. First, we analyzed the expression patterns of transcripts commonly associated with OG regulatory functions to describe their possible role once the maturation of the gonad is complete. The transcriptomic profiles of the optic gland of both sexes were compared with emphasis on the signaling pathways involved in the dimorphism of reproductive traits. Results suggest that in the OG of males, the reproductive condition (mated or non-mated) did not affect the general expression profile. In contrast, more differentially expressed genes were observed in females. In mated females, the mRNA metabolic process and the response to norepinephrine were enriched, suggesting a high cellular activity in preparation for the laying of the embryos. Whereas in egg-laying females, energetic and metabolic processes were the most represented, including the oxidation-reduction process. Finally, the gene expression patterns in senescence females suggest a physiological response to starvation as well as upregulation of genes involved retrotransposon activity. In conclusion, more substantial fluctuations in gene expression were observed in the optic glands of the fertilized females compared to the males. Such differences might be associated with the regulation of the egg-laying and the onset of senescence.

Bacteria in (vermi)composted organic wastes mostly survive when applied to an arable soil cultivated with wheat (Triticum sp. L.).

Composting and vermicomposting are an environmentally friendly way to reduce pathogens in organic wastes and generate a valuable product that provides nutrients for crops. However, how the bacterial community structure changes during these different processes and if the bacteria applied with the (vermi)composted products survive in an arable cultivated soil is still largely unknown. In this study, we monitored how the bacterial community structure changed during conditioning, composting with and without Eisenia fetida, and when the end-product was applied to arable soil cultivated with wheat Triticum sp. L. The organic wastes used were biosolid, cow manure, and a mixture of both. Large changes occurred in the relative abundance of some of the most abundant bacterial genera during conditioning, but the changes were much smaller during composting or vermicomposting. The bacterial community structure was significantly different in the organic wastes during conditioning and (vermi)composting but adding E. fetida had no significant effect on it. Changes in the relative abundance of the bacterial groups in the (vermi)composted waste applied to the arable soil cultivated with wheat were small, suggesting that most survived even after 140 days. As such, applying (vermi)composted organic wastes not only adds nutrients to a crop but also contributes to the survival of plant growth-promoting bacteria found in the (vermi)compost. However, putative human pathogens found in the biosolid also survived in the arable soil, and their relative abundance remained high but mixing the biosolid with cow manure reduced that risk. It was found that applying (vermi)composted organic wastes to an arable soil not only provides plant nutrients and adds bacteria with plant growth-promoting capacities, but some putative pathogens also survived.

Perceived discrimination and alcohol-related problems among Hispanic college students: The protective role of serious harm reduction behaviors.

The present study investigated the use of protective behavioral strategies (PBS) as moderators of the association between perceived discrimination and alcohol-related problems among Hispanic college students. Participants who were between 18 and 24 years of age (n = 379) completed self-report measures. The results showed that greater perceived discrimination was associated with less frequent PBS use, more problematic alcohol use, and more alcohol-related problems, while greater use of PBS types was associated with fewer alcohol-related problems. Furthermore, serious harm-reduction behaviors moderated the association between perceived discrimination and alcohol-related problems. The findings suggest that serious harm-reduction behaviors may protect against the negative effects of perceived discrimination on alcohol-related problems among Hispanic college students.

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity.

BACKGROUND: Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. METHODS: In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. RESULTS: Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. CONCLUSIONS: Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.

Analysis of primer gunshot residue particles by laser induced breakdown spectroscopy and laser ablation inductively coupled plasma mass spectrometry.

This study reports novel approaches for the detection of gunshot residues (GSR) from the hands of individuals using Laser-Induced Breakdown Spectroscopy (LIBS) and Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS). The methods' performance was evaluated using 159 GSR standard and authentic samples. Forty specimens generated from characterized microparticles were used as matrix-matched primer gunshot residue (pGSR) standards to examine the elemental profiles of leaded and lead-free residues, compared to SEM-EDS and solution-ICP-MS. Also, 119 authentic skin samples were analyzed to estimate error rates. Shooter samples were correctly classified into three categories based on their elemental composition (leaded, lead-free, or mixed pGSR). A total of 60 non-shooter samples were used to establish background thresholds and estimate specificity (93.4% for LA-ICP-MS and 100% for LIBS). All the authentic leaded items resulted in the detection of particle(s) with composition characteristic of pGSR (Pb-Ba-Sb), as observed by simultaneous elemental identification of target analytes at the exact ablation times and locations. When considering the pre-characterized elemental composition of these primers as the "ground truth", LA-ICP-MS resulted in 91.8% sensitivity (true positive rate), while LIBS resulted in 89.2% sensitivity. Particles containing Ba, Bi, Bi-Cu-K, and Cu-Ti-Zn were found in the lead-free residues. Identification of lead-free GSR proved more challenging as some of these elements are common in the environment, resulting in 85.2% sensitivity for LA-ICP-MS and 44.4% for LIBS. Overall accuracies of 94.9% and 88.2% were obtained for the LA-ICP-MS and LIBS sets, respectively. LA-ICP-MS provided an additional level of confidence in the results by its superior analytical capabilities, complementing the LIBS chemical profiles. The laser-based methods provide rapid chemical profiling and micro-spatial information of gunshot residue particles, with minimal destruction of the sample and high accuracy. Chemical mapping of 25 micro-regions per sample is possible in 2-10 minutes by LIBS and LA-ICP-MS, offering new tools for more comprehensive forensic case management and quick GSR screening in environmental and occupational sciences.

Determination of seventeen major and trace elements in new float glass standards for use in forensic comparisons using laser ablation inductively coupled plasma mass spectrometry.

Consensus concentration values for seventeen (17) major and trace elements typically present in soda-lime glass manufactured using the "float " process and used in the quantitative analysis and forensic comparison of glass samples were determined using laser ablation (LA) micro sampling coupled to inductively coupled plasma mass spectrometry (ICP-MS). This is the first reporting of the chemical characterization of a new set of float glass intended for use as matrix-matched calibration standards in the forensic analysis and comparison of glass by LA-ICP-MS using a standard test method (ASTM E2927-16e1). Three Corning Float Glass Standards (CFGS) were manufactured at low, medium, and high concentrations of 32 elements typically encountered in float glass samples as found in forensic casework. This work describes an international collaboration among seven (7) laboratories to evaluate the homogeneity of the three glass materials and reports the consensus concentrations values of 17 elements at three concentration levels. Eight (8) sets of independent results from LA-ICP-MS analysis using the standard test method of analysis and one set of micro-X-ray Fluorescence Spectrometry (µXRF) data (using method ASTM E2926-17) resulted in typically <3% relative standard deviation (RSD) within each lab and < 5% RSDs among all labs participating in the study for the concentration ranges using sampling spots between 50 µm - 100 µm in diameter. These results suggest that the new calibration standards are homogeneous for most elements at the small sampling volumes (~ 90 µm deep by ~80 µm in diameter) reported and show excellent agreement among the different participating labs. Consensus concentration values are determined using a previously reported calibration standard (FGS 2) and checked with a NIST 1831 SRM®. A collaboration with National Institute of Standards and Technology (NIST) scientists to certify these glasses as SRMs, including the certification of the quantitative analysis of the minor and trace element content, for future distribution by NIST is ongoing.

Antisaccades and memory-guided saccades in genetic generalized epilepsy and temporal lobe epilepsy.

OBJECTIVE: Oculomotor tasks can be used to measure volitional control of behavior sensitive to frontal dysfunction. This study aimed to examine the saccadic eye movement in Genetic Generalized Epilepsy (GGE) which could correlate with the abnormality of the frontal lobe or the thalamo-frontal network. METHODS: Twenty-one patients with GGE were compared with 22 patients with Temporal Lobe Epilepsy (TLE) and 39 healthy controls. Visual-guided saccades, Antisaccades, and Memory-guided saccades as oculomotor tasks were performed using a novel gaze-tracker designed for clinical practice use. RESULTS: Patients with epilepsy (either GEE or TLE) had similar latency, accuracy, and velocity in visual-guided saccades and memory-guided saccades. Patients with epilepsy had similar latencies and correct antisaccade number. However, healthy volunteers, matched by age, had faster responses and more accurate results than patients with epilepsy. CONCLUSIONS: Our investigations did not reveal differences between TLE and GGE patients' groups in visually guided saccades, antisaccades, and memory-guided saccades, thus suggesting that the frontal cortical mechanisms responsible for them are not explicitly impaired in patients with GGE.

Clinical, pathological and genetic spectrum in 89 cases of mitochondrial progressive external ophthalmoplegia.

BACKGROUND: Mitochondrial progressive external ophthalmoplegia (PEO) encompasses a broad spectrum of clinical and genetic disorders. We describe the phenotypic subtypes of PEO and its correlation with molecular defects and propose a diagnostic algorithm. METHODS: Retrospective analysis of the clinical, pathological and genetic features of 89 cases. RESULTS: Three main phenotypes were found: 'pure PEO' (42%), consisting of isolated palpebral ptosis with ophthalmoparesis; Kearns-Sayre syndrome (10%); and 'PEO plus', which associates extraocular symptoms, distinguishing the following subtypes: : myopathic (33%), bulbar (12%) and others (3%). Muscle biopsy was the most accurate test, showing mitochondrial changes in 95%. Genetic diagnosis was achieved in 96% of the patients. Single large-scale mitochondrial DNA (mtDNA) deletion was the most frequent finding (63%), followed by multiple mtDNA deletions (26%) due to mutations in TWNK (n=8), POLG (n=7), TK2 (n=6) or RRM2B (n=2) genes, and point mtDNA mutations (7%). Three new likely pathogenic mutations were identified in the TWNK and MT-TN genes. CONCLUSIONS: Phenotype-genotype correlations cannot be brought in mitochondrial PEO. Muscle biopsy should be the first step in the diagnostic flow of PEO when mitochondrial aetiology is suspected since it also enables the study of mtDNA rearrangements. If no mtDNA deletions are identified, whole mtDNA sequencing should be performed.

VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy.

Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.

The Nutrition Benefits Participation Gap: Barriers to Uptake of SNAP and WIC Among Latinx American Immigrant Families.

To examine nutrition benefit under-enrollment in Latinx American immigrant families, we administered a survey to 100 adults attending a NY Latinx American community serving organization. We used a logistic regression approach to analyze misinformation impact on enrollment, and examined non-enrollment explanations, among participants in whose families a child or pregnant or breastfeeding woman appeared SNAP- or WIC-eligible. Among households (N = 51) with ≥ 1 SNAP-eligible child, 49% had no child enrolled. Reasons included repercussion fears (e.g. payback obligation, military conscription, college aid ineligibility, child removal, non-citizen family member penalties), and logistical barriers. In multivariable regression models, having heard the rumor that SNAP/WIC participation makes unauthorized status family members vulnerable to being reported to the government was associated with an 85% lower enrollment rate (OR 0.15, CI 0.03, 0.94). Misinformation impedes nutrition benefit participation. A multi-level intervention is necessary to inform potential applicants and providers regarding eligibility criteria and erroneous rumors, along with an informed discussion of the risks versus benefits of using resources, especially as public charge criteria change.

Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.