Long-term olfactory loss post-COVID-19: Pathobiology and potential therapeutic strategies.

An acute loss of smell emerged as a striking symptom present in roughly half of the people infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in the early phases of the COVID-19 pandemic. In most COVID-19 patients, olfaction recovers over the course of a few weeks. However, a lasting partial or complete loss of smell, often associated with distorted olfactory perceptions termed parosmia, has emerged as a widespread problem impacting at least 5%-10% of those who experience anosmia due to COVID-19. Our inability to offer effective therapies to this hyposmic or anosmic population, comprising millions of patients, highlights an enormous unmet need for the medical system. Here, we summarize the current understanding of the pathobiology causing acute olfactory loss due to SARS-CoV-2 infection, focusing on how the virus interacts with the peripheral olfactory system, a major site of viral infection. We also explore the problem of long-COVID olfactory dysfunction, which may accompany other persistent systemic disorders collectively termed postacute sequelae of COVID-19. Specifically, we discuss an emerging model focused on unresolved immune cell activity driving ongoing dysfunction. Finally, we review current and future therapeutic approaches aimed at restoring olfactory function.

Olfactory neuroblastoma mimics molecular heterogeneity and lineage trajectories of small-cell lung cancer.

The olfactory epithelium undergoes neuronal regeneration from basal stem cells and is susceptible to olfactory neuroblastoma (ONB), a rare tumor of unclear origins. Employing alterations in Rb1/Trp53/Myc (RPM), we establish a genetically engineered mouse model of high-grade metastatic ONB exhibiting a NEUROD1+ immature neuronal phenotype. We demonstrate that globose basal cells (GBCs) are a permissive cell of origin for ONB and that ONBs exhibit cell fate heterogeneity that mimics normal GBC developmental trajectories. ASCL1 loss in RPM ONB leads to emergence of non-neuronal histopathologies, including a POU2F3+ microvillar-like state. Similar to small-cell lung cancer (SCLC), mouse and human ONBs exhibit mutually exclusive NEUROD1 and POU2F3-like states, an immune-cold tumor microenvironment, intratumoral cell fate heterogeneity comprising neuronal and non-neuronal lineages, and cell fate plasticity-evidenced by barcode-based lineage tracing and single-cell transcriptomics. Collectively, our findings highlight conserved similarities between ONB and neuroendocrine tumors with significant implications for ONB classification and treatment.

UV-C LED-induced cyclobutane pyrimidine dimer formation, lesion repair and mutagenesis in the biofilm-forming diatom, Navicula incerta.

The use of ultraviolet-C (UV-C) irradiation in marine biofouling control is a relatively new and potentially disruptive technology. This study examined effects of UV-C exposure on the biofilm-forming diatom, Navicula incerta. UV-C-induced mutations were identified via Illumina HiSeq. A de novo genome was assembled from control sequences and reads from UV-C-exposed treatments were mapped to this genome, with a quantitative estimate of mutagenesis then derived from the frequency of single nucleotide polymorphisms. UV-C exposure increased cyclobutane pyrimidine dimer (CPD) abundance with a direct correlation between lesion formation and fluency. Cellular repair mechanisms gradually reduced CPDs over time, with the highest UV-C fluence treatments having the fastest repair rates. Mutation abundances were, however, negatively correlated with CPD abundance suggesting that UV-C exposure may influence lesion repair. The threshold fluence for CPD formation exceeding CPD repair was >1.27 J cm-2. Fluences >2.54 J cm-2 were predicted to inhibit repair mechanisms. While UV-C holds considerable promise for marine antifouling, diatoms are just one, albeit an important, component of marine biofouling communities. Determining fluence thresholds for other representative taxa, highlighting the most resistant, would allow UV-C treatments to be specifically tuned to target biofouling organisms, whilst limiting environmental effects and the power requirement.

Safety and Feasibility of Steroid-Eluting Stent as a Bolster in Endoscopic Anterior Skull Base Reconstruction.

BACKGROUND: With a rising incidence of cerebrospinal fluid (CSF) leaks, endoscopic endonasal CSF leak repair is increasingly performed. Current approaches utilize a variety of materials including free mucosal grafts and vascularized flaps, but post-op leaks continue to be reported. Steroid-eluting bioabsorbable stents (SES) are used during functional endoscopic sinus surgery for chronic rhinosinusitis to reduce inflammation and scarring while maintaining patency of sinus ostia. OBJECTIVE: The aim of this study is to assess the feasibility of SES as a graft/flap bolster for endoscopic endonasal CSF leak repair. METHODS: This is a retrospective review of patients undergoing endoscopic endonasal CSF leak repair with SES placed as part of the bolster technique at a tertiary care center between January 2019 and May 2022. Age, sex, BMI, comorbid idiopathic intracranial hypertension, pathology, location of CSF leak, intraoperative CSF leak flow, reconstruction type, and presence of post-op CSF leak were recorded. RESULTS: Twelve patients (mean age 52, median BMI 30.9, 58% female) had SES placement as part of the bolster technique. The most common pathology was meningoencephalocele (75%). Reconstruction was performed with either a free mucosal graft (6), or a flap (6). No post-op CSF leaks occurred at a reconstruction site with a stent, and no known complications were reported. All sinusotomies were patent at the last follow-up visit. CONCLUSIONS: SES placement as an adjunct to graft and/or flap bolster appears to be safe and feasible during anterior skull base reconstruction and CSF leak repair providing longer term structural support and preserving sinus drainage patency.

Amphiphilic Balance: Effect of the Hydrophilic-Hydrophobic Ratio on Fouling-Release Surfaces.

This study demonstrated the importance of identifying the optimal balance of hydrophilic and hydrophobic moieties in amphiphilic coatings to achieve fouling-release (FR) performance that surpasses that of traditional hydrophobic marine coatings. While there have been many reports on fouling-release properties of amphiphilic surfaces, the offered understanding is often limited. Hence, this work is focused on further understanding of the amphiphilic surfaces. Poly(ethylene glycol) (PEG) and polydimethylsiloxane (PDMS) were used to create a series of noncross-linked amphiphilic additives that were then added to a hydrophobic-designed siloxane-polyurethane (SiPU) FR system. After being characterized by ATR-FTIR, XPS, contact angle analysis, and AFM, the FR performance was evaluated by using different marine organisms. The assessments showed that the closer the hydrophilic and hydrophobic moieties in a system reached a relatively equalized level, the more desirable the FR performance of the coating system became. A balanced ratio of hydrophilicity-hydrophobicity in the system at around 10-15 wt % of each component had the best FR performance and was comparable to or better than commercial FR coatings.

Diffusely Charged Polymeric Zwitterions as Loosely Hydrated Marine Antifouling Coatings.

Polymeric zwitterions exhibit exceptional fouling resistance through the formation of a strongly hydrated surface of immobilized water molecules. While being extensively tested for their performance in biomedical, membrane, and, to a lesser extent, marine environments, few studies have investigated how the molecular design of the zwitterion may enhance its performance. Furthermore, while theories of zwitterion antifouling mechanisms exist for molecular-scale foulant species (e.g., proteins and small molecules), it remains unclear how molecular-scale mechanisms influence the micro- and macroscopic interactions of relevance for marine applications. The present study addresses these gaps through the use of a modular zwitterion chemistry platform, which is characterized by a combination of surface-sensitive sum frequency generation (SFG) vibrational spectroscopy and marine assays. Zwitterions with increasingly delocalized cations demonstrate improved fouling resistance against the green alga Ulva linza. SFG spectra correlate well with the assay results, suggesting that the more diffuse charges exhibit greater surface hydration with more bound water molecules. Hence, the number of bound interfacial water molecules appears to be more influential in determining the marine antifouling activities of zwitterionic polymers than the binding strength of individual water molecules at the interface.

Polycomb repressive complex 2 regulates basal cell fate during adult olfactory neurogenesis.

Adult neurogenesis occurs in the mammalian olfactory epithelium to maintain populations of neurons that are vulnerable to injury yet essential for olfaction. Multipotent olfactory basal stem cells are activated by damage, although mechanisms regulating lineage decisions are not understood. Using mouse lesion models, we focused on defining the role of Polycomb repressive complexes (PRCs) in olfactory neurogenesis. PRC2 has a well-established role in developing tissues, orchestrating transcriptional programs via chromatin modification. PRC2 proteins are expressed in olfactory globose basal cells (GBCs) and nascent neurons. Conditional PRC2 loss perturbs lesion-induced neuron production, accompanied by altered histone modifications and misexpression of lineage-specific transcription factors in GBCs. De-repression of Sox9 in PRC2-mutant GBCs is accompanied by increased Bowman's gland production, defining an unrecognized role for PRC2 in regulating gland versus neuron cell fate. Our findings support a model for PRC2-dependent mechanisms promoting sensory neuronal differentiation in an adult neurogenic niche.

Preparation of Human Olfactory Epithelial Biopsies for Downstream Analysis.

The olfactory mucosa, lining a portion of the nasal cavity, houses the primary olfactory sensory neurons responsible for odor transduction, along with supporting cell populations. Tremendous advances have come from studying the peripheral olfactory system in animal models, especially the mouse. However, acquired human olfactory disorders lack effective therapies, and many of these conditions involve pathology in the olfactory mucosa. Thus, the ability to obtain human olfactory biopsy samples from subjects with olfactory dysfunction, or controls, may be of value. Here, we describe established techniques for collecting olfactory tissue from human subjects and preparing samples for downstream assays such as immunohistochemistry, flow cytometry, single-cell RNA-sequencing, or chromatin studies.

Deconstructing Olfactory Epithelium Developmental Pathways in Olfactory Neuroblastoma.

Olfactory neuroblastoma is a rare tumor arising from the olfactory cleft region of the nasal cavity. Because of the low incidence of this tumor, as well as an absence of established cell lines and murine models, understanding the mechanisms driving olfactory neuroblastoma pathobiology has been challenging. Here, we sought to apply advances from research on the human olfactory epithelial neurogenic niche, along with new biocomputational approaches, to better understand the cellular and molecular factors in low- and high-grade olfactory neuroblastoma and how specific transcriptomic markers may predict prognosis. We analyzed a total of 19 olfactory neuroblastoma samples with available bulk RNA-sequencing and survival data, along with 10 samples from normal olfactory epithelium. A bulk RNA-sequencing deconvolution model identified a significant increase in globose basal cell (GBC) and CD8 T-cell identities in high-grade tumors (GBC from ∼0% to 8%, CD8 T cell from 0.7% to 2.2%), and significant decreases in mature neuronal, Bowman's gland, and olfactory ensheathing programs, in high-grade tumors (mature neuronal from 3.7% to ∼0%, Bowman's gland from 18.6% to 10.5%, olfactory ensheathing from 3.4% to 1.1%). Trajectory analysis identified potential regulatory pathways in proliferative olfactory neuroblastoma cells, including PRC2, which was validated by immunofluorescence staining. Survival analysis guided by gene expression in bulk RNA-sequencing data identified favorable prognostic markers such as SOX9, S100B, and PLP1 expression. Significance: Our analyses provide a basis for additional research on olfactory neuroblastoma management, as well as identification of potential new prognostic markers.

Characterizing Olfactory Dysfunction in Patients with Unilateral Cleft Lip Nasal Deformities.

Background: Unilateral cleft lip nasal deformity (uCLND) is associated with olfactory dysfunction, but the underlying etiology remains poorly understood. Objective: To investigate the etiology of uCLND-associated olfactory dysfunction using clinical, computational, and histologic assessments. Methods: Inclusion criteria: uCLND patients >16 years undergoing septorhinoplasty. Exclusion criteria: prior septoplasty or rhinoplasty, pregnancy, sinusitis. Measured outcomes: patient-reported scores, rhinomanometry, smell identification and threshold tests, computational fluid dynamics (CFD) airflow simulations, and histologic analysis of olfactory epithelium. Results: Five uCLND subjects were included: 18-23 years, three male and two female, four left-sided cleft and one right-sided cleft. All subjects reported moderate to severe nasal obstruction. Smell identification and threshold tests showed varying degrees of hyposmia. Nasal resistance was higher on the cleft side versus noncleft side measured by rhinomanometry (median 3.85 Pa-s/mL, interquartile range [IQR] = 21.96, versus 0.90 Pa-s/mL, IQR = 5.17) and CFD (median 1.04 Pa-s/mL, IQR = 0.94 vs. 0.11 Pa-s/mL, IQR = 0.12). Unilateral olfaction varied widely and was dependent on unilateral percentage olfactory airflow. Biopsies revealed intact olfactory neuroepithelium. Conclusions: uCLND-associated olfactory dysfunction appears to be primarily conductive in etiology and highly susceptible to variations in nasal anatomy. Clinical Trial Registration number: NCT04150783.

Nitroxide-Containing Amphiphilic Random Terpolymers for Marine Antifouling and Fouling-Release Coatings.

Two types of amphiphilic random terpolymers, poly(ethylene glycol methyl ether methacrylate)-ran-poly(2,2,6,6-tetramethylpiperidinyloxy methacrylate)-ran-poly(polydimethyl siloxane methacrylate) (PEGMEMA-r-PTMA-r-PDMSMA), were synthesized and evaluated for antifouling (AF) and fouling-release (FR) properties using diverse marine fouling organisms. In the first stage of production, the two respective precursor amine terpolymers containing (2,2,6,6-tetramethyl-4-piperidyl methacrylate) units (PEGMEMA-r-PTMPM-r-PDMSMA) were synthesized by atom transfer radical polymerization using various comonomer ratios and two initiators: alkyl halide and fluoroalkyl halide. In the second stage, these were selectively oxidized to introduce nitroxide radical functionalities. Finally, the terpolymers were incorporated into a PDMS host matrix to create coatings. AF and FR properties were examined using the alga Ulva linza, the barnacle Balanus improvisus, and the tubeworm Ficopomatus enigmaticus. The effects of comonomer ratios on surface properties and fouling assay results for each set of coatings are discussed in detail. There were marked differences in the effectiveness of these systems against the different fouling organisms. The terpolymers had distinct advantages over monopolymeric systems across the different organisms, and the nonfluorinated PEG and nitroxide combination was identified as the most effective formulation against B. improvisus and F. enigmaticus.

Persistent post-COVID-19 smell loss is associated with immune cell infiltration and altered gene expression in olfactory epithelium.

SARS-CoV-2 causes profound changes in the sense of smell, including total smell loss. Although these alterations are often transient, many patients with COVID-19 exhibit olfactory dysfunction that lasts months to years. Although animal and human autopsy studies have suggested mechanisms driving acute anosmia, it remains unclear how SARS-CoV-2 causes persistent smell loss in a subset of patients. To address this question, we analyzed olfactory epithelial samples collected from 24 biopsies, including from nine patients with objectively quantified long-term smell loss after COVID-19. This biopsy-based approach revealed a diffuse infiltrate of T cells expressing interferon-γ and a shift in myeloid cell population composition, including enrichment of CD207+ dendritic cells and depletion of anti-inflammatory M2 macrophages. Despite the absence of detectable SARS-CoV-2 RNA or protein, gene expression in the barrier supporting cells of the olfactory epithelium, termed sustentacular cells, appeared to reflect a response to ongoing inflammatory signaling, which was accompanied by a reduction in the number of olfactory sensory neurons relative to olfactory epithelial sustentacular cells. These findings indicate that T cell-mediated inflammation persists in the olfactory epithelium long after SARS-CoV-2 has been eliminated from the tissue, suggesting a mechanism for long-term post-COVID-19 smell loss.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

Studying the Effect of Pre-Polymer Composition and Incorporation of Surface-Modifying Amphiphilic Additives on the Fouling-Release Performance of Amphiphilic Siloxane-Polyurethane Coatings.

Combining amphiphilic fouling-release (FR) coatings with the surface-active nature of amphiphilic additives can improve the antifouling/fouling-release (AF/FR) properties needed to offer broad-spectrum resistance to marine biofoulants. This work is focused on further tuning the amphiphilic character of a previously developed amphiphilic siloxane-polyurethane (SiPU) coating by varying the amount of PDMS and PEG in the base system. Furthermore, surface-modifying amphiphilic additives (SMAAs) were incorporated into these amphiphilic FR SiPU coatings in varying amounts. ATR-FTIR, contact angle and surface energy measurements, and AFM were performed to assess changes in surface composition, wettability, and morphology. AF/FR properties were evaluated using laboratory biological assays involving Cellulophaga lytica, Navicula incerta, Ulva linza, Amphibalanus amphitrite, and Geukensia demissa. The surfaces of these coatings varied significantly upon changes in PDMS and PEG content in the coating matrix, as well as with changes in SMAA incorporation. AF/FR properties were also significantly changed, with formulations containing the highest amounts of SMAA showing very high removal properties compared to other experimental formulations, in some cases better than that of commercial standard FR coatings.

Olfactory dysfunction and COVID-19.

Here, we provide an overview of olfactory dysfunction associated with COVID-19. We provide background regarding the organization and function of the peripheral olfactory system. A review of the relevant literature on anosmia and parosmia due to infection with SARS-CoV-2, the virus causing COVID-19, is provided. Specific attention is focused on possible mechanisms by which the virus may interact with and damage the cell populations of peripheral olfactory system. Evidence from human studies as well as animal models is considered. Finally, we discuss current recommendations for evaluation and management of patients with persistent post-COVID olfactory dysfunction, as well as possible future research directions.

Investigation of N-Substituted Morpholine Structures in an Amphiphilic PDMS-Based Antifouling and Fouling-Release Coating.

Biofouling is a major disruptive process affecting the fuel efficiency and durability of maritime vessel coatings. Previous research has shown that amphiphilic coatings consisting of a siloxane backbone functionalized with hydrophilic moieties are effective marine antifouling and fouling-release materials. Poly(ethylene glycol) (PEG) has been the primary hydrophilic component used in such systems. Recently, the morpholine group has emerged as a promising compact alternative in antifouling membranes but is yet to be studied against marine foulants. In this work, the use of morpholine moieties to generate amphiphilicity in a poly(dimethylsiloxane) (PDMS)-based antifouling and fouling-release coating was explored. Two separate coating sets were investigated. The first set examined the incorporation of an N-substituted morpholine amine, and while these coatings showed promising fouling-release properties for Ulva linza, they had unusually high settlement of spores compared to controls. Based on those results, a second set of materials was synthesized using an N-substituted morpholine amide to probe the source of the high settlement and was found to significantly improve antifouling performance. Both coating sets included PEG controls with varying lengths to compare the viability of the morpholine structures as alternative hydrophilic groups. Surfaces were evaluated through a combination of bubble contact angle goniometry, profilometry, X-ray photoelectron spectroscopy (XPS), and marine bioassays against two soft fouling species, U. linza and Navicula incerta, known to have different adhesion characteristics.

Persistent post-COVID-19 smell loss is associated with inflammatory infiltration and altered olfactory epithelial gene expression.

Most human subjects infected by SARS-CoV-2 report an acute alteration in their sense of smell, and more than 25% of COVID patients report lasting olfactory dysfunction. While animal studies and human autopsy tissues have suggested mechanisms underlying acute loss of smell, the pathophysiology that underlies persistent smell loss remains unclear. Here we combine objective measurements of smell loss in patients suffering from post-acute sequelae of SARS-CoV-2 infection (PASC) with single cell sequencing and histology of the olfactory epithelium (OE). This approach reveals that the OE of patients with persistent smell loss harbors a diffuse infiltrate of T cells expressing interferon-gamma; gene expression in sustentacular cells appears to reflect a response to inflammatory signaling, which is accompanied by a reduction in the number of olfactory sensory neurons relative to support cells. These data identify a persistent epithelial inflammatory process associated with PASC, and suggests mechanisms through which this T cell-mediated inflammation alters the sense of smell.

Low Fouling Polysulfobetaines with Variable Hydrophobic Content.

Amphiphilic polymer coatings combining hydrophilic elements, in particular zwitterionic groups, and hydrophobic elements comprise a promising strategy to decrease biofouling. However, the influence of the content of the hydrophobic component in zwitterionic coatings on the interfacial molecular reorganization dynamics and the anti-fouling performance is not well understood. Therefore, coatings of amphiphilic copolymers of sulfobetaine methacrylate 3-[N-2'-(methacryloyloxy)ethyl-N,N-dimethyl]-ammonio propane-1-sulfonate (SPE) are prepared which contain increasing amounts of hydrophobic n-butyl methacrylate (BMA). Their fouling resistance is compared to that of their homopolymers PSPE and PBMA. The photo-crosslinked coatings form hydrogel films with a hydrophilic surface. Fouling by the proteins fibrinogen and lysozyme as well as by the diatom Navicula perminuta and the green algae Ulva linza is assessed in laboratory assays. While biofouling is strongly reduced by all zwitterionic coatings, the best fouling resistance is obtained for the amphiphilic copolymers. Also in preliminary field tests, the anti-fouling performance of the amphiphilic copolymer films is superior to that of both homopolymers. When the coatings are exposed to a marine environment, the reduced susceptibility to silt incorporation, in particular compared to the most hydrophilic polyzwitterion PSPE, likely contributes to the improved fouling resistance.

Amphiphilic Nitroxide-Bearing Siloxane-Based Block Copolymer Coatings for Enhanced Marine Fouling Release.

The buildup of organic matter and organisms on surfaces exposed to marine environments, known as biofouling, is a disruptive and costly process affecting maritime operations. Previous research has identified some of the surface characteristics particularly suited to the creation of antifouling and fouling-release surfaces, but there remains room for improvement against both macrofouling and microfouling organisms. Characterization of their adhesives has shown that many rely on oxidative chemistries. In this work, we explore the incorporation of the stable radical 2,2,6,6-tetramethylpipiderin-1-oxyl (TEMPO) as a component in an amphiphilic block copolymer system to act as an inhibitor for marine cements, disrupting adhesion of macrofouling organisms. Using polystyrene-b-poly(dimethylsiloxane-r-vinylmethysiloxane) block copolymers, pendent vinyl groups were functionalized with TEMPO and poly(ethylene glycol) to construct an amphiphilic material with redox active character. The antifouling and fouling-release performance of these materials was investigated through settlement and removal assays of three model fouling organisms and correlated to surface structure and chemistry. Surfaces showed significant antifouling character and fouling-release performance was increased substantially toward barnacles by the incorporation of stable radicals, indicating their potential for marine antifouling applications.

Surface modifying amphiphilic additives and their effect on the fouling-release performance of siloxane-polyurethane coatings.

In this work, surface-modifying amphiphilic additives (SMAAs) were synthesized via hydrosilylation using various polymethylhydrosiloxanes (PMHS) and allyl-terminated polyethylene glycol monomethyl ethers (APEG) of varying molecular weights. The additives synthesized were incorporated into a hydrophobic, self-stratifying siloxane-polyurethane (SiPU) coating system to produce an amphiphilic surface. Contact angle experiments and atomic force microscopy (AFM), in a dry and hydrated state, were performed to assess changes in surface wettability and morphology. The antifouling and fouling-release (AF/FR) performances were evaluated by performing laboratory biological assays using the marine bacterium Cellulophaga lytica, the microalga Navicula incerta, the macroalga Ulva linza, the barnacle Amphibalanus amphitrite, and the marine mussel, Geukensia demissa. Several of the formulations showed improved AF/FR performance vs the base SiPU and performed better than some of the commercial standard marine coatings. Formulations containing SMAAs with a low grafting density of relatively high molecular weight PEG chains showed the best performance overall.