Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections

Application of single-molecule switching nanoscopy (SMSN) beyond the coverslip surface poses substantial challenges due to sample-induced aberrations that distort and blur single-molecule emission patterns. We combined active shaping of point spread functions and efficient adaptive optics to enable robust 3D-SMSN imaging within tissues. This development allowed us to image through 30-μm-thick brain sections to visualize and reconstruct the morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer's disease

Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity

Phthalate and novel plasticizer concentrations in food items from U.S. fast food chains: a preliminary analysis

BACKGROUND: Fast food consumption is associated with biomarkers of ortho-phthalates exposures. However, the chemical content of fast food is unknown; certain ortho-phthalates (i.e., di-n-butyl phthalate (DnBP) and di(2-ethylhexyl) phthalate (DEHP)) have been phased out and replaced with other plasticizers (e.g., dioctyl terephthalate (DEHT)). OBJECTIVE: We conducted a preliminary study to examine ortho-phthalate and replacement plasticizer concentrations in foods and food handling gloves from U.S. fast food restaurants. METHODS: We obtained hamburgers, fries, chicken nuggets, chicken burritos, cheese pizza (n = 64 food samples) and gloves (n = 3) from restaurants and analyzed them for 11 chemicals using gas chromatography mass spectrometry. RESULTS: We found DEHT at the highest concentrations in both foods (n = 19; median = 2510 µg/kg; max = 12,400 µg/kg) and gloves (n = 3; range: 28-37% by weight). We detected DnBP and DEHP in 81% and 70% of food samples, respectively. Median DEHT concentrations were significantly higher in burritos than hamburgers (6000 µg/kg vs. 2200 µg/kg; p \u3c 0.0001); DEHT was not detected in fries. Cheese pizza had the lowest levels of most chemicals. SIGNIFICANCE: To our knowledge, these are the first measurements of DEHT in food. Our preliminary findings suggest that ortho-phthalates remain ubiquitous and replacement plasticizers may be abundant in fast food meals. IMPACT STATEMENT: A selection of popular fast food items sampled in this study contain detectable levels of replacement plasticizers and concerning ortho-phthalates. In addition, food handling gloves contain replacement plasticizers, which may be a source of food contamination. These results, if confirmed, may inform individual and regulatory exposure reduction strategies

Heavy Metal Bioaccumulation in Peruvian Food and Medicinal Products

To better query regional sources of metal(loid) exposure in an under-communicated region, available scientific literature from 50 national universities (undergraduate and graduate theses and dissertations), peer-reviewed journals, and reports published in Spanish and English were synthesized with a focus on metal(loid) bioaccumulation in Peruvian food and medicinal products utilized locally. The study considered 16 metal(loid)s that are known to exert toxic impacts on humans (Hg, Al, Sb, As, Ba, Be, Cd, Cr, Sn, Ni, Ag, Pb, Se, Tl, Ti, and U). A total of 1907 individual analyses contained within 231 scientific publications largely conducted by Peruvian universities were analyzed. These analyses encompassed 239 reported species classified into five main food/medicinal groups—plants, fish, macroinvertebrates and mollusks, mammals, and “others” category. Our benchmark for comparison was the World Health Organization (Codex Alimentarius) standards. The organisms most frequently investigated included plants such as asparagus, corn, cacao, and rice; fish varieties like trout, tuna, and catfish; macroinvertebrates and mollusks including crab and shrimp; mammals such as alpaca, cow, chicken eggs, and milk; and other categories represented by propolis, honey, lichen, and edible frog. Bioaccumulation-related research increased from 2 to more than 25 publications per year between 2006 and 2022. The results indicate that Peruvian food and natural medicinal products can have dangerous levels of metal(loid)s, which can cause health problems for consumers. Many common and uncommon food/medicinal products and harmful metals identified in this analysis are not regulated on the WHO’s advisory lists, suggesting the urgent need for stronger regulations to ensure public safety. In general, Cd and Pb are the metals that violated WHO standards the most, although commonly non-WHO regulated metals such as Hg, Al, As, Cr, and Ni are also a concern. Metal concentrations found in Peru are on many occasions much higher than what has been reported elsewhere. We conclude that determining the safety of food/medicinal products is challenging due to varying metal concentrations that are influenced not only by metal type but also geographical location. Given the scarcity of research findings in many regions of Peru, urgent attention is required to address this critical knowledge gap and implement effective regulatory measures to protect public health

Lassa Virus Targeting of Anterior Uvea and Endothelium of Cornea and Conjunctiva in Eye of Guinea Pig Model

Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors

Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in Ifnar-/- mice.

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/β receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations