Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb.

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.

Regulation of the probability of mouse odorant receptor gene choice.

Each olfactory sensory neuron (OSN) in mouse chooses one of 1,200 odorant receptor (OR) genes for expression. OR genes are chosen for expression by greatly varying numbers of OSNs. The mechanisms that regulate the probability of OR gene choice remain unclear. Here, we have applied the NanoString platform of fluorescent barcodes and digital readout to measure RNA levels of 577 OR genes in a single reaction, with probes designed against coding sequences. In an inbred mouse strain with a targeted deletion in the P element, we find that this element regulates OR gene choice differentially across its cluster of 24 OR genes. Importantly, the fold changes of NanoString counts in ΔP or ΔH mice are in very close agreement with the fold changes of cell counts, determined by in situ hybridization. Thus, the P and H elements regulate the probability of OR gene choice, not OR transcript level per OSN.

Expert curation of the human and mouse olfactory receptor gene repertoires identifies conserved coding regions split across two exons.

BACKGROUND: Olfactory receptor (OR) genes are the largest multi-gene family in the mammalian genome, with 874 in human and 1483 loci in mouse (including pseudogenes). The expansion of the OR gene repertoire has occurred through numerous duplication events followed by diversification, resulting in a large number of highly similar paralogous genes. These characteristics have made the annotation of the complete OR gene repertoire a complex task. Most OR genes have been predicted in silico and are typically annotated as intronless coding sequences. RESULTS: Here we have developed an expert curation pipeline to analyse and annotate every OR gene in the human and mouse reference genomes. By combining evidence from structural features, evolutionary conservation and experimental data, we have unified the annotation of these gene families, and have systematically determined the protein-coding potential of each locus. We have defined the non-coding regions of many OR genes, enabling us to generate full-length transcript models. We found that 13 human and 41 mouse OR loci have coding sequences that are split across two exons. These split OR genes are conserved across mammals, and are expressed at the same level as protein-coding OR genes with an intronless coding region. Our findings challenge the long-standing and widespread notion that the coding region of a vertebrate OR gene is contained within a single exon. CONCLUSIONS: This work provides the most comprehensive curation effort of the human and mouse OR gene repertoires to date. The complete annotation has been integrated into the GENCODE reference gene set, for immediate availability to the research community.

The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation.

Spermatogonial stem cells (SSCs) are adult stem cells that are slowly cycling and self-renewing. The pool of SSCs generates very large numbers of male gametes throughout the life of the individual. SSCs can be cultured in vitro for long periods of time, and established SSC lines can be manipulated genetically. Upon transplantation into the testes of infertile mice, long-term cultured mouse SSCs can differentiate into fertile spermatozoa, which can give rise to live offspring. Here, we show that the testicular soma of mice with a conditional knockout (conKO) in the X-linked gene Tsc22d3 supports spermatogenesis and germline transmission from cultured mouse SSCs upon transplantation. Infertile males were produced by crossing homozygous Tsc22d3 floxed females with homozygous ROSA26-Cre males. We obtained 96 live offspring from six long-term cultured SSC lines with the aid of intracytoplasmic sperm injection. We advocate the further optimization of Tsc22d3-conKO males as recipients for testis transplantation of SSC lines.

Exclusive transmission of the embryonic stem cell-derived genome through the mouse germline.

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell-derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26-Cre males. This cross produces males that are sterile due to a complete cell-autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell-derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene-targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non-ES cell-derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326-333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

The extremely broad odorant response profile of mouse olfactory sensory neurons expressing the odorant receptor MOR256-17 includes trace amine-associated receptor ligands.

The mouse olfactory system employs ~1100 G-protein-coupled odorant receptors (ORs). Each mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and the expressed OR determines the odorant response properties of the OSN. The broadest odorant response profile thus far demonstrated in native mouse OSNs is for OSNs that express the OR gene SR1 (also known as Olfr124 and MOR256-3). Here we showed that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256-17 (also known as Olfr15 and OR3) is even broader than that of OSNs expressing SR1. We investigated the electrophysiological properties of green fluorescent protein (GFP)+ OSNs in a MOR256-17-IRES-tauGFP gene-targeted mouse strain, in parallel with GFP+ OSNs in the SR1-IRES-tauGFP gene-targeted mouse strain that we previously reported. Of 35 single chemical compounds belonging to distinct structural classes, MOR256-17+ OSNs responded to 31 chemicals, compared with 10 for SR1+ OSNs. The 10 compounds that activated SR1+ OSNs also activated MOR256-17+ OSNs. Interestingly, MOR256-17+ OSNs were activated by three amines (cyclohexylamine, isopenthylamine, and phenylethylamine) that are typically viewed as ligands for chemosensory neurons in the main olfactory epithelium that express trace amine-associated receptor genes, a family of 15 genes encoding G-protein-coupled receptors unrelated in sequence to ORs. We did not observe differences in membrane properties, indicating that the differences in odorant response profiles between the two OSN populations were due to the expressed OR. MOR256-17+ OSNs appear to be at one extreme of odorant responsiveness among populations of OSNs expressing distinct OR genes in the mouse.

A family of nonclassical class I MHC genes contributes to ultrasensitive chemodetection by mouse vomeronasal sensory neurons.

The mouse vomeronasal organ (VNO) has a pivotal role in chemical communication. The vomeronasal sensory neuroepithelium consists of distinct populations of vomeronasal sensory neurons (VSNs). A subset of VSNs, with cell bodies in the basal part of the basal layer, coexpress Vmn2r G-protein-coupled receptor genes with H2-Mv genes, a family of nine nonclassical class I major histocompatibility complex genes. The in vivo, physiological roles of the H2-Mv gene family remain mysterious more than a decade after the discovery of combinatorial H2-Mv gene expression in VSNs. Here, we have taken a genetic approach and have deleted the 530 kb cluster of H2-Mv genes in the mouse germline by chromosome engineering. Homozygous mutant mice (ΔH2Mv mice) are viable and fertile. There are no major anatomical defects in their VNO and accessory olfactory bulb (AOB). Their VSNs can be stimulated with chemostimuli (peptides and proteins) to the same maximum responses as VSNs of wild-type mice, but require much higher concentrations. This physiological phenotype is displayed at the single-cell level and is cell autonomous: single V2rf2-expressing VSNs, which normally coexpress H2-Mv genes, display a decreased sensitivity to a peptide ligand in ΔH2Mv mice, whereas single V2r1b-expressing VSNs, which do not coexpress H2-Mv genes, show normal sensitivity to a peptide ligand in ΔH2Mv mice. Consistent with the greatly decreased VSN sensitivity, ΔH2Mv mice display pronounced deficits in aggressive and sexual behaviors. Thus, H2-Mv genes are not absolutely essential for the generation of physiological responses, but are required for ultrasensitive chemodetection by a subset of VSNs.

Statistical analysis of differential gene expression relative to a fold change threshold on NanoString data of mouse odorant receptor genes.

BACKGROUND: A challenge in gene expression studies is the reliable identification of differentially expressed genes. In many high-throughput studies, genes are accepted as differentially expressed only if they satisfy simultaneously a p value criterion and a fold change criterion. A statistical method, TREAT, has been developed for microarray data to assess formally if fold changes are significantly higher than a predefined threshold. We have recently applied the NanoString digital platform to study expression of mouse odorant receptor genes, which form with 1,200 members the largest gene family in the mouse genome. Our objectives are, on these data, to decrease false discoveries when formally assessing the genes relative to a fold change threshold, and to provide a guided selection in the choice of this threshold. RESULTS: Statistical tests have been developed for microarray data to identify genes that are differentially expressed relative to a fold change threshold. Here we report that another approach, which we refer to as tTREAT, is more appropriate for our NanoString data, where false discoveries lead to costly and time-consuming follow-up experiments. Methods that we refer to as tTREAT2 and the running fold change model improve the performance of the statistical tests by protecting or selecting the fold change threshold more objectively. We show the benefits on simulated and real data. CONCLUSIONS: Gene-wise statistical analyses of gene expression data, for which the significance relative to a fold change threshold is important, give reproducible and reliable results on NanoString data of mouse odorant receptor genes. Because it can be difficult to set in advance a fold change threshold that is meaningful for the available data, we developed methods that enable a better choice (thus reducing false discoveries and/or missed genes) or avoid this choice altogether. This set of tools may be useful for the analysis of other types of gene expression data.

Temporal patterns of odorant receptor gene expression in adult and aged mice.

In the mouse, the sense of smell relies predominantly on the expression of ~1200 odorant receptor (OR) genes in the main olfactory epithelium (MOE). Each mature olfactory sensory neuron (OSN) in the MOE is thought to express just one of these OR genes; conversely, an OR gene is expressed in thousands to tens of thousands of OSNs per mouse. Here, we have characterized temporal patterns of OR gene expression in a cohort of inbred C57BL6/N mice from the Aged Rodent Colonies of the National Institute on Aging. We applied the NanoString multiplex platform to quantify RNA abundance for 531 OR genes in whole olfactory mucosa (WOM) tissue samples. The five study groups were females aged 2, 6, 12, 18, and 31 months (mo). We classified the 531 temporal patterns using a step-down quadratic regression method for time course analysis. The majority of OR genes (58.4%) are classified as flat: there is no significant difference from a horizontal line within this time window. There are 32.8% of OR genes with a downward profile, 7.2% with an upward profile, and 1.7% with a convex or concave profile. But the magnitude of these decreases and increases tends to be small: only 4.3% of OR genes are differentially expressed (DE) at 31 mo compared to 2 mo. Interestingly, the variances of NanoString counts for individual OR genes are homogeneous among the age groups. Our analyses of these 15,930 OR gene expression data of C57BL6/N mice that were raised and housed under well-controlled conditions indicate that OR gene expression at the MOE level is intrinsically stable.

Protocol for postmortem bedside endoscopic procedure to sample human respiratory and olfactory cleft mucosa, olfactory bulbs, and frontal lobe.

We present a protocol for the rapid postmortem bedside procurement of selected tissue samples using an endoscopic endonasal surgical technique that we adapted from skull base surgery. We describe steps for the postmortem collection of blood, cerebrospinal fluid, a nasopharyngeal swab, and tissue samples; the clean-up procedure; and the initial processing and storage of the samples. This protocol was validated with tissue samples procured postmortem from COVID-19 patients and can be applied in another emerging infectious disease. For complete details on the use and execution of this protocol, please refer to Khan et al. (2021)1 and Khan et al. (2022).2.

It's a trap?! Escape from an ancient, ancestral sex chromosome system and implication of Foxl2 as the putative primary sex-determining gene in a lizard (Anguimorpha; Shinisauridae).

Although sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally to genetically influenced. In vertebrates, genetic sex determination is typically accomplished with sex chromosomes. Groups like mammals maintain conserved sex chromosome systems, while sex chromosomes in most vertebrate clades are not conserved across similar evolutionary timescales. One group inferred to have an evolutionarily stable mode of sex determination is Anguimorpha, a clade of charismatic taxa including monitor lizards, Gila monsters, and crocodile lizards. The common ancestor of extant anguimorphs possessed a ZW system that has been retained across the clade. However, the sex chromosome system in the endangered, monotypic family of crocodile lizards (Shinisauridae) has remained elusive. Here, we analyze genomic data to demonstrate that Shinisaurus has replaced the ancestral anguimorph ZW system on LG7 with a novel ZW system on LG3. The linkage group, LG3, corresponds to chromosome 9 in chicken, and this is the first documented use of this syntenic block as a sex chromosome in amniotes. Additionally, this ~1 Mb region harbors approximately 10 genes, including a duplication of the sex-determining transcription factor, Foxl2, critical for the determination and maintenance of sexual differentiation in vertebrates, and thus a putative primary sex-determining gene for Shinisaurus.

Visualising SARS-CoV-2 infection of the lung in deceased COVID-19 patients.

BACKGROUND: SARS-CoV-2 is a single-stranded positive-sense RNA virus. Several negative-sense SARS-CoV-2 RNA species, both full-length genomic and subgenomic, are produced transiently during viral replication. Methodologies for rigorously characterising cell tropism and visualising ongoing viral replication at single-cell resolution in histological sections are needed to assess the virological and pathological phenotypes of future SARS-CoV-2 variants. We aimed to provide a robust methodology for examining the human lung, the major target organ of this RNA virus. METHODS: A prospective cohort study took place at the University Hospitals Leuven in Leuven, Belgium. Lung samples were procured postmortem from 22 patients who died from or with COVID-19. Tissue sections were fluorescently stained with the ultrasensitive single-molecule RNA in situ hybridisation platform of RNAscope combined with immunohistochemistry followed by confocal imaging. FINDINGS: We visualised perinuclear RNAscope signal for negative-sense SARS-CoV-2 RNA species in ciliated cells of the bronchiolar epithelium of a patient who died with COVID-19 in the hyperacute phase of the infection, and in ciliated cells of a primary culture of human airway epithelium that had been infected experimentally with SARS-CoV-2. In patients who died between 5 and 13 days after diagnosis of the infection, we detected RNAscope signal for positive-sense but not for negative-sense SARS-CoV-2 RNA species in pneumocytes, macrophages, and among debris in the alveoli. SARS-CoV-2 RNA levels decreased after a disease course of 2-3 weeks, concomitant with a histopathological change from exudative to fibroproliferative diffuse alveolar damage. Taken together, our confocal images illustrate the complexities stemming from traditional approaches in the literature to characterise cell tropism and visualise ongoing viral replication solely by the surrogate parameters of nucleocapsid-immunoreactive signal or in situ hybridisation for positive-sense SARS-CoV-2 RNA species. INTERPRETATION: Confocal imaging of human lung sections stained fluorescently with commercially available RNAscope probes for negative-sense SARS-CoV-2 RNA species enables the visualisation of viral replication at single-cell resolution during the acute phase of the infection in COVID-19. This methodology will be valuable for research on future SARS-CoV-2 variants and other respiratory viruses. FUNDING: Max Planck Society, Coronafonds UZ/KU Leuven, European Society for Organ Transplantation.

It's a Trap?! Escape from an ancient, ancestral sex chromosome system and implication of Foxl2 as the putative primary sex determining gene in a lizard (Anguimorpha; Shinisauridae).

Although sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally- to genetically-influenced. In vertebrates, genetic sex determination is typically accomplished with sex chromosomes. Groups like mammals maintain conserved sex chromosome systems, while sex chromosomes in most vertebrate clades aren't conserved across similar evolutionary timescales. One group inferred to have an evolutionarily stable mode of sex determination is Anguimorpha, a clade of charismatic taxa including: monitor lizards, Gila monsters, and crocodile lizards. The common ancestor of extant anguimorphs possessed a ZW system that has been retained across the clade. However, the sex chromosome system in the endangered, monotypic family of crocodile lizards (Shinisauridae) has remained elusive. Here, we analyze genomic data to demonstrate that Shinisaurus has replaced the ancestral anguimorph ZW system on LG7 chromosome with a novel ZW system on LG3. The linkage group LG3 corresponds to chromosome 9 in chicken, and this is the first documented use of this syntenic block as a sex chromosome in amniotes. Additionally, this ~1Mb region harbors approximately 10 genes, including a duplication of the sex-determining transcription factor, Foxl2-critical for the determination and maintenance of sexual differentiation in vertebrates, and thus a putative primary sex determining gene for Shinisaurus.

Lung epithelial and myeloid innate immunity in influenza-associated or COVID-19-associated pulmonary aspergillosis: an observational study.

BACKGROUND: Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) affect about 15% of critically ill patients with influenza or COVID-19, respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality, but data on their pathophysiology are scarce. We aimed to explore the role of lung epithelial and myeloid innate immunity in patients with IAPA or CAPA. METHODS: In this observational study, we retrospectively recruited patients who had been admitted to the intensive care unit (ICU) of University Hospitals Leuven, Belgium, requiring non-invasive or invasive ventilation because of severe influenza or COVID-19, with or without aspergillosis, between Jan 1, 2011, and March 31, 2021, whose bronchoalveolar lavage samples were available at the hospital biobank. Additionally, biobanked in vivo tracheobronchial biopsy samples from patients with IAPA or CAPA and invasive Aspergillus tracheobronchitis admitted to ICUs requiring invasive ventilation between the same dates were collected from University Hospitals Leuven, Hospital Network Antwerp (Belgium), and Amiens-Picardie University Hospital (France). We did nCounter gene expression analysis of 755 genes linked to myeloid innate immunity and protein analysis of 47 cytokines, chemokines, and growth factors on the bronchoalveolar lavage samples. Gene expression data were used to infer cell fractions by use of CIBERSORTx, to perform hypergeometric enrichment pathway analysis and gene set enrichment analysis, and to calculate pathway module scores for the IL-1ß, TNF-α, type I IFN, and type II IFN (IFNγ) pathways. We did RNAScope targeting influenza virus or SARS-CoV-2 RNA and GeoMx spatial transcriptomics on the tracheobronchial biopsy samples. FINDINGS: Biobanked bronchoalveolar lavage samples were retrieved from 166 eligible patients, of whom 40 had IAPA, 52 had influenza without aspergillosis, 33 had CAPA, and 41 had COVID-19 without aspergillosis. We did nCounter gene expression analysis on bronchoalveolar lavage samples from 134 patients, protein analysis on samples from 162 patients, and both types of analysis on samples from 130 patients. We performed RNAScope and spatial transcriptomics on the tracheobronchial biopsy samples from two patients with IAPA plus invasive Aspergillus tracheobronchitis and two patients with CAPA plus invasive Aspergillus tracheobronchitis. We observed a downregulation of genes associated with antifungal effector functions in patients with IAPA and, to a lesser extent, in patients with CAPA. We found a downregulated expression of several genes encoding proteins with functions in the opsonisation, recognition, and killing of conidia in patients with IAPA versus influenza only and in patients with CAPA versus COVID-19 only. Several genes related to LC3-associated phagocytosis, autophagy, or both were differentially expressed. Patients with CAPA had significantly lower neutrophil cell fractions than did patients with COVID-19 only. Patients with IAPA or CAPA had downregulated IFNγ signalling compared with patients with influenza only or COVID-19 only, respectively. The concentrations of several fibrosis-related growth factors were significantly elevated in the bronchoalveolar lavage fluid from patients with IAPA versus influenza only and from patients with CAPA versus COVID-19 only. In one patient with CAPA, we visualised an active or very recent SARS-CoV-2 infection disrupting the epithelial barrier, facilitating tissue-invasive aspergillosis. INTERPRETATION: Our results reveal a three-level breach in antifungal immunity in IAPA and CAPA, affecting the integrity of the epithelial barrier, the capacity to phagocytise and kill Aspergillus spores, and the ability to destroy Aspergillus hyphae, which is mainly mediated by neutrophils. The potential of adjuvant IFNγ in the treatment of IAPA and CAPA should be investigated. FUNDING: Research Foundation Flanders, Coronafonds, the Max Planck Society, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, "la Caixa" Foundation, and Horizon 2020.

Anatomical barriers against SARS-CoV-2 neuroinvasion at vulnerable interfaces visualized in deceased COVID-19 patients.

Can SARS-CoV-2 hitchhike on the olfactory projection and take a direct and short route from the nose into the brain? We reasoned that the neurotropic or neuroinvasive capacity of the virus, if it exists, should be most easily detectable in individuals who died in an acute phase of the infection. Here, we applied a postmortem bedside surgical procedure for the rapid procurement of tissue, blood, and cerebrospinal fluid samples from deceased COVID-19 patients infected with the Delta, Omicron BA.1, or Omicron BA.2 variants. Confocal imaging of sections stained with fluorescence RNAscope and immunohistochemistry afforded the light-microscopic visualization of extracellular SARS-CoV-2 virions in tissues. We failed to find evidence for viral invasion of the parenchyma of the olfactory bulb and the frontal lobe of the brain. Instead, we identified anatomical barriers at vulnerable interfaces, exemplified by perineurial olfactory nerve fibroblasts enwrapping olfactory axon fascicles in the lamina propria of the olfactory mucosa.

A Prosthetically Guided Technique for Cast Post-and-Core Fabrication.

Despite it being an older, conventional production method, the cast metal post and core is still often considered the best option for the restoration of severely damaged teeth. The direct technique for fabrication of cast post-and-core patterns, however, can pose challenges due to the inefficiencies and guesswork involved in creating an appropriate form and dimension for the core segment. This article presents an enhanced technique for cast post-and-core fabrication in reference to the desired dimensions of the final restoration. As the authors demonstrate, the procedure involves creation of an accurate and passive pattern of each post space. Bis-acrylic composite resin is then injected into a putty impression of the idealized wax-up and seated on the prepared post patterns. A preparation of the abutments is then performed by creating the cores according to the desired dimensions of the final restoration. The major advantages of this technique include a more efficient workflow and a reduction in the number of adjustments needed after insertion.

Danger perception and stress response through an olfactory sensor for the bacterial metabolite hydrogen sulfide.

The olfactory system serves a critical function as a danger detection system to trigger defense responses essential for survival. The cellular and molecular mechanisms that drive such defenses in mammals are incompletely understood. Here, we have discovered an ultrasensitive olfactory sensor for the highly poisonous bacterial metabolite hydrogen sulfide (H2S) in mice. An atypical class of sensory neurons in the main olfactory epithelium, the type B cells, is activated by both H2S and low O2. These two stimuli trigger, respectively, Cnga2- and Trpc2-signaling pathways, which operate in separate subcellular compartments, the cilia and the dendritic knob. This activation drives essential defensive responses: elevation of the stress hormone ACTH, stress-related self-grooming behavior, and conditioned place avoidance. Our findings identify a previously unknown signaling paradigm in mammalian olfaction and define type B cells as chemosensory neurons that integrate distinct danger inputs from the external environment with appropriate defense outputs.

Investigation of the Cellular Destination of Fluorescently Labeled Carbon Nanohorns in Cultured Cells.

The high surface area, facile functionalization, and biocompatibility of carbon nanohorns (CNHs) make them attractive for many applications, including drug delivery. The cellular destination of nanomaterials dictates both the therapeutic application and the potential toxicity. Identifying the uptake mechanism is challenging as several endocytic pathways have been identified that facilitate cellular entry. Here, the cellular uptake of fluorescently labeled CNHs was assessed by utilizing quantitative cell-based assays to determine the factors influencing how internalization occurs and the destinations they reach in HeLa cells. Cell viability assays suggest that about 80% of the cells remained viable even at the highest concentration of 20 µg/mL exposure to CNHs. Uptake studies revealed that when pulse-chase conditions were applied, CNHs were seen to be localized both at the cell periphery and in a juxtanuclear pattern inside HeLa cells, in the latter case colocalizing with the lysosomal marker LAMP1. RNA interference studies, using a panel of RNA tools to individually deplete key molecules associated with the endocytic machinery, failed to block the internalization of CNHs into cells, suggesting that multiple mechanisms of endocytosis are used by this particle type.

Comparison of Label and Laboratory Sodium Values in Popular Sodium-Contributing Foods in the United States.

BACKGROUND: Nutrition labels are important tools for consumers and for supporting public health strategies. Recent, published comparison of label and laboratory sodium values for US foods, and differences by brand type (national or private-label) or source (store or restaurant [fast-food and sit-down]) is unavailable. OBJECTIVE: The objective was to compare label and laboratory values for sodium and related nutrients (ie, total sugars, total fat, and saturated fat) in popular, sodium-contributing foods, and examine whether there are differences by brand type, and source. DESIGN: During 2010 to 2014, the Nutrient Data Laboratory of the US Department of Agriculture collected 3,432 samples nationwide of 125 foods, combined one or more samples of the same food (henceforth referred to as composites), and chemically analyzed them. For this comparative post hoc analysis, the Nutrient Data Laboratory linked laboratory values for 1,390 composites (consisting of one or more samples of the same food) of 114 foods to corresponding label or website (restaurant) nutrient values. MAIN OUTCOME MEASURES: Label and laboratory values and their ratio for each composite, for each of the four nutrients (sodium, total fat, total sugars, and saturated fat). STATISTICAL ANALYSES PERFORMED: Nutrient Data Laboratory analysis determined the ratio of laboratory to label value for each composite, and categorized them into six groups: ≥141%, 121% to 140%, 101% to 120%, 81% to 100%, 61% to 80%, and ≤60%. For sodium, the Nutrient Data Laboratory analysis determined the distribution of the ratios by food, food category, brand type, and source. RESULTS: For sodium, 5% of the composites had ratios of laboratory to label values >120% and 14% had ratios ≤80%. Twenty-two percent of private-label brand composites had ratios ≤80%, compared with 12% of national brands. Only 3% of store composites had ratios >120% compared with 11% of restaurant composites. Ratios ≤80% were more prevalent among sit-down restaurants (37%) compared with fast-food restaurants (9%). CONCLUSIONS: This study shows that a majority of label and laboratory values sampled agree and underdeclaration of label values is limited. However, there is some disagreement. Periodic monitoring of the nutrient content of foods through laboratory analyses establishes validity of the food labels and helps identify foods and food categories where the label and laboratory values do not compare well, and hence may need laboratory analyses to support accuracy of food composition data.

A transcriptomic atlas of mammalian olfactory mucosae reveals an evolutionary influence on food odor detection in humans.

The mammalian olfactory system displays species-specific adaptations to different ecological niches. To investigate the evolutionary dynamics of olfactory sensory neuron (OSN) subtypes across mammalian evolution, we applied RNA sequencing of whole olfactory mucosa samples from mouse, rat, dog, marmoset, macaque, and human. We find that OSN subtypes, representative of all known mouse chemosensory receptor gene families, are present in all analyzed species. Further, we show that OSN subtypes expressing canonical olfactory receptors are distributed across a large dynamic range and that homologous subtypes can be either highly abundant across all species or species/order specific. Highly abundant mouse and human OSN subtypes detect odorants with similar sensory profiles and sense ecologically relevant odorants, such as mouse semiochemicals or human key food odorants. Together, our results allow for a better understanding of the evolution of mammalian olfaction in mammals and provide insights into the possible functions of highly abundant OSN subtypes.