Traumatic brain injury (TBI) causes structural damage and functional impairment in the visual system, often with retinal ganglion cell (RGC) degeneration occurring without visual symptoms. RGC degeneration is associated with reduced retinal blood-flow, however, it is not known whether reductions in perfusion precede or are secondary to neurodegeneration.We conducted a prospective observational single-center case series. Patients were included if they were admitted to the hospital after acute TBI and underwent ophthalmic clinical examination, including optical coherence tomography (OCT) and OCT angiography (OCTA) acutely and at follow-up. Ganglion cell layer thickness (GCL) thickness, vascular density in the superficial vascular plexus (SVP), and intermediate capillary plexus (ICP) were quantified.P < 0.0001).Twenty-one patients aged 20 to 65 years (mean = 38 years) including 16 men and 5 women were examined less than 14 days after moderate to severe TBI, and again after 2 to 6 months. Macular structure and perfusion were normal at baseline in all patients. Visual function was abnormal at baseline in three patients and subsequent neurodegeneration and loss of perfusion corresponded to baseline visual function abnormalities. Nine patients (43%) had reduced macular GCL thickness at follow up. Perfusion in the SVP strongly associated with local GCL thickness. The strongest association of the SVP metrics was the sum of vessel density (In cases of reduced visual function after TBI, macular perfusion remained normal until reductions in GCL thickness occurred, indicating that perfusion changes were secondary to local GCL loss. A related imaging modality, OCT angiography (OCTA), demonstrates flow in the retinal microstructure.–,–Retinal perfusion is reduced in relation to cerebral perfusion with retinal perfusion mirroring cerebral perfusion in healthy patients.–Retinal perfusion is reduced in local vascular disease, such as anterior ischemic optic neuropathy, where optic nerve head hypoperfusion precedes and causes RGC degeneration.We therefore assessed GCL thickness and retinal vascular changes using OCT and OCTA in subjects after acute TBI to assess whether vascular changes preceded or followed neuronal degeneration, hypothesizing that reduction in perfusion would precede and predict neurodegeneration.Prospective data were collected on patients with TBI as part of a service evaluation registered with the Clinical Governance Department, and data analyses for research were approved by the Health Research Authority and by the Research and Development Department at University Hospitals Birmingham NHS Foundation Trust. Control patients were recruited under: Defining Outcome Measures in Ocular Inflammatory Disease: 14/EM/1163; Ophthalmic and Neurocognitive Assessment in the Management of Critically Ill Patients: 19/YH/0113; and Eye Assessments in Traumatic Brain Injury: 20/WM/0178.Inclusion criteria for the wider service evaluation dataset were adult patients diagnosed with acute TBI between October 2020 and October 2021 and able to cooperate with OCT examination. Patients were included in the retrospective analysis if they had OCTA <14 days after injury (acute) and at follow-up between 6 weeks and 6 months after injury. The acute time point was chosen to occur before RGC loss is detectable on OCT, and the follow-up time point after optic atrophy occurs in TON.Patients were excluded from the data analysis if they had any direct penetrating or nonpenetrating injury to the eye, any pre-existing optic nerve or retinal pathology, or if they were unable to cooperate with the examination. Only patients with complete datasets for both eyes were included.Control patients were included if they had no history of eye or brain disease and were within 5 years of age of an unmatched patient with TBI included in the study.Data collected in addition to OCT and OCTA scans included age, sex, cause of trauma, neuroradiological findings, severity of TBI, ophthalmic examination findings, presence of visual symptoms at acute visit, and time to acute and follow-up assessment.TBI severity was based on the US Department of Defense (DoD) classification.Table 1OCT and OCTA images were acquired using the SPECTRALIS Heidelberg OCT2 table-top module . Each OCT volume consisted of the manufacturer's “Posterior Pole” protocol to acquire volumetric retinal scans in 61 horizontal B-scans averaged over at least 9 frames in a 30 degrees × 25 degrees fovea-centered volume. Each OCTA volume consisted of 521 B-scans with an interscan distance of 1.79 µm nominal spacing in a 6 × 6 mm total area. Seven repeated OCT B-scans at each tissue location were used by the full-spectrum probabilistic OCTA algorithm to determine the presence or absence of flow at each voxel.Measurements of GCL thickness in the macula within the Early Treatment of Diabetic Retinopathy Study (ETDRS) grid areas were obtained using manufacturer's software . RGC loss after TBI between baseline and follow-up was classified on OCT as: “no change” where GCL loss was ≤3 µm,OCTA data were exported as .E2E files for further analysis using manufacturer's software . The onboard volumetric PAR algorithm (“3D PAR vessel strong”) that performs 3D vessel shape estimation in the volume and only operates to remove their projection tails was used to remove projection artifacts. Serial follow-up OCT and OCTA scans were co-registered on the basis of the fundus image ensuring that exactly the same area is imaged on repeat scans. As perfusion is measured at a single point in time and averaged across the temporal window of the scan time, a variability measure is not provided.The macula was divided into the ETDRS grid areas and perfusion analysis within individual sectors compared with GCL thickness. The ETDRS grid areas are C0 , S1 (inner and superior), S2 (outer and superior), T1 , T2 , I1 (inner and inferior), I2 (outer and inferior), N1 , and N2 . Software-calculated perfusion metrics were exported in the superficial vascular plexus, intermediate capillary plexus, and deep capillary plexus : Mean, sum, probability (prob), skeletonized vascular density (skeleton), perfusion density, vessel length density, vessel enhanced perfusion density (vessel enhanced), and large vessel masked flow sum (LVMS) and probability (LVMP).These metrics evaluate: mean – expected probability of OCTA signal for a pixel in the vascular layer; sum – amount of OCTA signal within the vascular layer; prob – probability that OCTA signal exists in the vascular layer; skeleton – density of vessel center lines; vessel enhanced – scaled version of probability output to suppress noise while preserving small vessels; LVMS/LVMP – sum/probability output, respectively, with large vessels removed from the analysis; perfusion density – measured in a binarized image; and vessel length density – derived from skeletonized vascular density using manufacturer's formula.P < 0.05 was considered statistically significant. Measures from right and left eyes were modeled as repeated measures on the same subject at each time point . Partial squared eta (η2p) was used to assess the proportion of variability in the dependent variable explained by individual independent variables in the model. In general, eta squared is calculated the same way as R squared  and has values between 0 and 1 (similar to R squared). As more variables are added, each individual eta squared will reduce as each variable will explain the some of the variance. We have used partial eta squared where any variation explained by other variables is removed from the denominator. It is generally considered that η2 = 0.01 indicates a small effect; η2 = 0.06 indicates a medium effect; and η2 = 0.14 indicates a large effect. Control, excluded, and included patients were compared using the t-test for continuous data and chi-squared test for proportions.The results were analyzed using R software. Perfusion was modeled as a dependent variable using linear regression, GCL thickness as a covariate, and time point after injury, and eye and retinal area (in ETDRS grid) as predictor variables. A value of Raw data were generated at and are owned by the University Hospitals Birmingham NHS Foundation Trust. Derived data supporting the findings of this study are available from the corresponding author on reasonable request, subject to a data transfer agreement.P = <0.001), had a higher percentage of men, overall lower severity of TBI (P = <0.001), and higher proportion resulting from sports injury (P = <0.001) when compared to the patients who were included in the study.Patient characteristics at entry into the study are shown in In our cohort, isolated hemorrhagic contusions were present in 4 of 21 (19%) subjects, skull/orbital or cervical vertebral fractures in 8 of 21 (39%), and intracranial hemorrhages, such as subdural (SDH), subarachnoid (SAH), and intraparenchymal/cerebral hemorrhages (ICH) were present in 9 of 21 43%; see . OverallA normal clinical examination, with visual acuity of 20/20 or better, normal color vision, pupils, and confrontational fields, was present in 86% patients (18/21) at the acute assessment stage see .Comparison among the 21 included participants and the wider screened cohort within the service evaluation and the control group did not reveal a significant difference in GCL thickness or retinal perfusion  acutely One patient had a unilateral severe TON, which was clinically apparent in the acute visit as a relative afferent pupillary defect (RAPD) and unilateral no light perception (NLP). Two patients had homonymous field loss to confrontation on acute assessment. In all 3 of 21 (14%) patients with abnormal visual assessment in the acute visit, there was severe GCL thinning at follow-up with a mean GCL thickness change of −20 µm for the patient with TON, and –8 µm for the patients with homonymous loss . A furthReductions in retinal perfusion in the SVP mirrored GCL thickness loss, corresponding to baseline abnormalities of visual function where these existed . SVP mea2p was close to zero across all measures demonstrating a weaker relationship  with primary neural and vascular structural injury.,,The eye and brain share embryological tissue origins. Retinal microvascular changes reflect cerebrovascular changes in neurodegenerative diseases, such as stroke and dementia, suggesting that retinal perfusion may mirror cerebral perfusion after TBI.–,,,GCL thickness is used as a marker for optic nerve health with GCL thinning the final common and irreversible result of optic nerve injury.–,,–Although TON is reported in 0.5% to 8% of patients with TBI, OCT and field abnormalities may be detectable in up to 53% of patients with TBI even without detectable deficits in visual acuity.RGCs are situated in the inner retina, closely associated with the SVP, whereas the ICP and DCP bracket the inner nuclear layer.The weaker association between GCL and the skeletonized perfusion metrics could be explained by the fact that skeletonization estimates a single vessel line in the center of a vessel, reducing each vessel to a single pixel width. Changes in retinal perfusion causing changes in vessel caliber will therefore not be detected on skeletonized analysis. The SVP has larger vessels as well as capillaries. For large vessels, the software occasionally maps two parallel lines on either side of the vessel, instead of one line in the center of the vessel. Software can also have difficulty skeletonizing capillary vessels in the presence of larger vessels, which is significant for the SVP where there are both large and small vessels. Skeletonized vessel length density may therefore be less reliable in the SVP than the ICP. The skeleton metric is also sensitive to scan quality, working poorly in the presence of artifacts.This study has several limitations. It was conducted as a single center retrospective review of patients included in a prospective clinical study. A larger prospective study could confirm the results, although the strength of the observed associations suggests that these are robust. Our inclusion criteria required patients to be able to cooperate with OCT imaging within 14 days of injury, and although 5 patients in our cohort had severe TBI, it would have excluded patients with more severe injury and marked vascular dysregulation who remained in intensive care. We were unable to relate the retinal findings to cerebral perfusion, which would be of interest particularly in severely injured patients. We also only examined two relatively broad time points designed to capture images before and after GCL loss had occurred and it would be of interest to conduct more frequent assessments in the first 12 weeks after injury.We show that retinal GCL thickness and SVP density are normal after acute TBI, even in the context of acute reductions in visual function. Reduction in retinal perfusion after TBI follows rather than precedes GCL loss.


Demonstrably, auxin-activated proton pumps, AGPs, Ca2+ channels, and auxin efflux “PIN” proteins, embedded in the plasma membrane, combine to generate cytosolic Ca2+ oscillations that ultimately regulate oscillatory growth: Hechtian adhesion of the plasma membrane to the cell wall and auxin-activated proton pumps trigger the release of dynamic Ca2+ stored in periplasmic AGP monolayers. These four major components represent a molecular PINball machine a strong visual metaphor that also recognises auxin efflux “PIN” proteins as an essential component. Proton “pinballs” dissociate Ca2+ ions bound by paired glucuronic acid residues of AGP glycomodules, hence reassessing the role of proton pumps. It shifts the prevalent paradigm away from the recalcitrant “acid growth” theory that proposes direct action on cell wall properties, with an alternative explanation that connects proton pumps to Ca2+ signalling with dynamic Ca2+ storage by AGPs, auxin transport by auxin-efflux PIN proteins and Ca2+ channels. The extensive Ca2+ signalling literature of plants ignores arabinogalactan proteins (AGPs). Such scepticism leads us to reconsider the validity of the universal growth oscillator proposed here with some exceptions that involve marine plants and perhaps the most complex stress test, stomatal regulation.Since Darwin’s “Power of Movement in Plants” the precise mechanism of oscillatory plant growth remains elusive. Hence the search continues for the hypothetical growth oscillator that regulates a huge range of growth phenomena ranging from circumnutation to pollen tube tip growth and stomatal movements. Oscillators are essentially simple devices with few components. A universal growth oscillator with only four major components became apparent recently with the discovery of a missing component, notably arabinogalactan glycoproteins (AGPs) that store dynamic Ca Pairs of kidney-shaped guard cells form stomatal pores that respond to ambient conditions by opening and closing and thus allow a trade-off between water retention and CO2 entry. A critical balance depends on the osmotic generation of turgor pressure and the elasticity of guard cell walls with anisotropic reinforcement. Guard cell turgor depends on the regulated influx and efflux of specific osmolytes. Opening and closing are distinct processes; both depend on Ca2+ signalling. However, a simple model remains elusive. Even after 50 years, the extensive literature shows that major problems remain stubbornly unresolved. This implies that a major piece of the puzzle is missing. Certainly, the glaring omission of AGPs from the current discussion is a strong hint of their possible relevance, especially considering their location and Ca2+ binding properties. The following discussion outlines how AGPs may contribute to the Ca2+ homeostasis that underlies the mechanical dynamics of stomatal function. This may resolve the apparent paradox of Ca2+ required for both opening and closing stomata [About four hundred million years ago, bryophytes evolved an upright sporophyte with a terminal sporogenous capsule that enhanced aerial spore dissemination but created the opposing demands of water retention while permitting carbon dioxide entry. The development of a waterproof cuticle saved water but restricted CO stomata ,2,3,4.2+ homeostasis, which was quite unanticipated. The Plant Kingdom has recruited reverse proton gradients; a novel extrapolation of Mitchell’s proton pump integrates auxin-controlled growth with cell surface glycoproteins and Ca2+ signalling as a major regulator of plant growth , and protein X-Ray diffraction by Perutz and Kendrew. Peter Mitchell’s discovery of chemiosmosis and the proton motif force generated by proton gradients across a membrane is one of the great Nobel Prize winners [t growth . This co2+. The Venn diagram emphasises common attributes shared by the four membrane components that are uniquely linked by their common function in Ca2+ homeostasis.Auxin efflux PIN proteins transport auxin.Auxin activates proton pump to generate protons.2+ from AGP-Ca2+.Protons release Ca2+ channels supply cytosolic Ca2+.Open CaA Venn diagram: four components embedded in the plasma membrane generate cytosolic Ca2+-binding sites [2+ stores of animal cells, plants locate their dynamic Ca2+ storage at the most strategic point of entry, the plasma membrane. The release of dynamic Ca2+ then identifies a novel function of proton pump activity.Cell surface arabinogalactan glycoproteins (AGPs) were for many years with only modest evidence  considerng sites . Further2+-dependent Ca2+ oscillator [2+ gradient; this suggested Ca2+ influx could originate from external stores in the cell wall [2+ and the cell surface Ca2+ oscillator explained its crucial role in pollen tube growth. The wide ramifications include not only tip growth of pollen tubes but numerous other plant processes such as phyllotaxis and even stomatal movements [Another centre of activity involved National Service at Number Two Radio School, RAF Yatesbury, where two years as an instructor in radio theory, with a focus on oscillators, provided the essential backdrop to biological oscillators. This knowledge was later critical in identifying the AGP-Cacillator . Over 25ell wall . Howeverell wall  became sovements .2+ homeostasis that regulates plant growth and differentiation thus exemplifying the parsimony of nature  released to the cytosol by inositol triphosphate IP3 of the Ca2+-signalling phosphoinositide (PI) pathway [2+ homeostasis: Indeed, it is now clear that plants store dynamic Ca2+ externally as periplasmic AGP-Ca2+ rather than in the ER. One hardly expects immediate acceptance of this model. Time sifts. Paradigms shift. Indeed our inference extrapolates Peter Mitchell’s chemiosmotic model that couples proton gradients across mitochondrial membranes with ATP generation [2+ homeostasis and growth. Somehow, plants have bypassed the enormous complexities of hormonal regulation in animals with an elegant strategy that regulates plant growth by unifying an auxin-activated proton pump with AGPs and Ca2+ homeostasis. Sydney Ringer’s serendipitous discovery of Catraction  was follmination . These d pathway . Many co pathway  reminiscneration   of the Arabidopsis genome create significant genetic redundancy and therefore a challenge to functional analysis.2+ hypothesis by two groups with substantial contributions to AGP structural chemistry generated multiple mutants that lack AGP glucuronic acid, the Dupree group at Cambridge UK, and the Showalter group at Athens Ohio. The genetic redundancy of glucuronosyl transferases that add GLcA to AGP polysaccharides involved complementary approaches to the generation of multiple GlcAT gene knockouts: Firstly, T-DNA insertion lines involving Ti plasmid transformation mediated by Agrobacterium tumefaciens generated Arabidopsis mutants in four glucuronosyl transferases  [A critical test of the AGP-Ca and -E) . Secondly, similar Arabidopsis mutants were generated by the CRISPRCas9 multiplexing approach for GlcAT14A, GlcAT14B, and GlcAT14C . 2+ homeostasis. This was consistent with the greatly decreased glucuronidation and Ca2+-binding capacity of AGPs isolated from leaves and roots of glcat14a/b/d mutants [2+ transients in roots with attenuated Ca2+ wave propagation. Significantly, exogenous Ca2+ suppressed/rescued these AGP glucuronidation mutant phenotypes.A comprehensive and detailed study of plant growth by the Dupree group showed that multiple glucuronosyl knockouts severely impaired Ca mutants , while tA parallel study by the Showalter group focussed on sexual reproduction and the role of multiple glucuronosyl transferase (glcat) mutants in Arabidopsis. Over many years significant contributions to AGP biology by the Showalter group have demonstrated the widespread involvement of AGPs encoded by 85 genes and 11 GLCAT genes. Their current work demonstrates that multiple glcat mutants significantly impair sexual reproduction, particularly pollen development, polytube block, and successful fertilisation that requires cytoplasmic calcium oscillations in synergid cells after physical contact with the pollen tube tip followed by normal embryo development in Arabidopsis.Taken together, the Dupree and Showalter groups corroborate the pinball model and support a unified theory of plant growth based on the properties of membrane glycoproteins.2+ homeostasis, therefore Glucuronic-bound Ca2+ is the essential source of cytosolic Ca2+. The novel idea of a Ca2+ store strategically located at the surface of the plant plasma membrane was previously overlooked because Mother Nature keeps some of her best secrets well hidden.Multiple glucuronosyl transferase knockouts severely impair Ca2+-binding centre.In a conventional pinball machine, pinball trajectories are largely random. Thus, although a strong visual metaphor a molecular pinball machine  has limi2+ has been apparent for many years [2+. While Vanneste and Friml concluded [2+ signalling is poorly understood”, Dindas and colleagues confirmed [2+-mediated metabolic regulation. An alternative explanation postulates complex auxin-activated Ca2+ channels [2+ channel. While they demonstrate Ca2+ influx within seconds of IAA application, it completely ignores the source of dynamic Ca2+ bound by cell surface AGP-Ca2+ stores, hence overlooking the direct role of the proton pump. Although a connection between auxin and Cany years , both auny years  with a concluded  that “thonfirmed  a close channels : Auxin  is of pThe proton pump acts as a central hub regulated by virtually all factors known to regulate plant growth, hormones, and classical plant hormones  generati2+ from AGP-Ca2+ and also generates the membrane potential. Thus AGP-Ca2+ dissociation initiates cytosolic Ca2+ oscillations while a large membrane potential enables K+ influx increasing cytosolic osmolyte levels including malate derived from starch. These Ca2+ oscillations provide the cytosolic Ca2+ that triggers vesicle exocytosis [2+ is evidenced by the over 200 EF-hand Ca2+-binding proteins as key transducers mediating Ca2+ action encoded in the Arabidopsis thaliana genome [In turgid guard cells, the proton pump activated by auxin releases Caocytosis ,40. Indea genome . + osmolyte via the specific K+ efflux channel GORK [2+ oscillations that regulate osmolyte influx and efflux respectively; increased cytosolic Ca2+ opens stomata [+ efflux channels and convert malate to starch. Such low demands presumably reflect the much weaker Ca2+ oscillations hence a low level of metabolic activity. This resolves the paradox, depicted in In flaccid cells, osmotic stress and dehydration generate abscisic acid, which triggers stomatal closure  by inactnel GORK  and recy stomata ,45. On tThe coastal town of Brighton afforded access to a wide range of ecological habitats including chalk downland, freshwater, estuarine, marine, and saltmarsh environments. These included the Lewes Brooks where salt ingress from the adjacent tidal river Ouse formed a clear ecological gradient. 2+ binding role of classical AGPs [2+ homeostasis regulated by a molecular pinball machine in the plasma membrane [2+ binding were of great interest, particularly in seawater species of flowering plants where high Na+ could disrupt Ca2+ binding. This was an evolutionary conundrum faced by flowering plants seeking to escape drought through the return to an oceanic abundance. Indeed, evolutionary adaptation to high salt has occurred in a family of seagrasses represented by Zostera marina [Ulva lactuca is also adapted to a marine environment but 90% of Chlorophyta are freshwater species; thus Ulva probably migrated from fresh to seawater very much earlier than Zostera [2+-binding [These early influences, which included summers living with a farm family and working on a mixed dairy/arable farm, led to a curiosity about natural processes and a career in biochemistry. On retirement joining the faculty at Sussex University as a visiting professor, Dean Timothy Flower’s interest in saltmarsh ecology stimulated my approach to salt stress and led to the discovery of upregulated AGP secretion in cultured cells . Elucidacal AGPs  that ledmembrane ,47,48,49a marina ,22. It c Zostera , yet qui-binding . Thus itWhile highly glycosylated classical AGPs are the most abundant in cell extracts, a small minority of AGPs with just a few arabinogalactan glycomodules are designated as non-classical; such minimal glycosylation directs them into the Golgi secretion pathway targeted to the plasma membrane . Their f2+ signalling [This review recapitulates recent significant progress in AGP biology represented by the molecular pinball machine of the plasma membrane that unifies proton pumps, AGPs, and Cagnalling .2+ can both open and close stomata, with AGPs as the missing link and source of cytosolic Ca2+. Arguably, the proton motive force controls both the opening and closing of stomata. Thus positive and negative regulation of the proton pump involves an auxin accelerator and an abscisic acid brake. That simple analogy explains how low Ca2+ levels close stomata, emphasising elegant control of both water loss and carbon dioxide gain.Here, we presented a critical test of the pinball hypothesis and suggested how it resolves the paradox that Ca2+ homeostasis and its related tropisms. It has taken many years to appreciate the versatility of these cell surface glycoproteins.Recent work validates the pinball hypothesis and is a suitable closure to a research career extending over 60 years initiated by the discovery of hydroxyproline-rich cell wall proteins  in 1960.


Did adults’ use of electronic nicotine delivery systems (ENDS) and cigarette smoking behaviors change after the Center for Tobacco Products (CTP) of the US Food and Drug Administration prioritized enforcement efforts against fruit-flavored cartridge ENDS in February 2020?This nationally representative US cohort study found a significant decrease in fruit-flavored cartridge ENDS use among adults in 2020; no differences were found in cigarette cessation or relapse rates between those who used ENDS that were vs were not targeted by the CTP.Findings provide no indication that the CTP’s 2020 targeted ENDS enforcement priority was associated with reduced smoking cessation or increased smoking relapse among adults who used ENDS. This cohort study evaluates the use of electronic nicotine delivery systems (ENDS) and cigarette smoking after prioritized enforcement efforts against the use of fruit-flavored nicotine cartridges. The Center for Tobacco Products (CTP) of the US Food and Drug Administration prioritized its enforcement efforts against non–tobacco-, non–menthol- (fruit-) flavored cartridge electronic nicotine delivery systems (ENDS) in February 2020.To evaluate adults’ use of ENDS and cigarette smoking following the CTP’s prioritized enforcement efforts against fruit-flavored cartridge ENDS.In this population-based, nationally representative US cohort study, data were collected from the Population Assessment of Tobacco and Health Study from December 2018 to November 2019 (hereafter referred to as 2019) and/or from September 2020 to December 2020 . Adults (aged ≥21 years) who used ENDS in the past 30 days and smoked cigarettes in the past 30 days or quit smoking cigarettes in the past year (n = 3173) were evaluated. Data were analyzed from January 1, 2022, to May 2, 2023.ENDS flavor-device combinations used.Outcome measures were cross-sectional prevalence of ENDS flavor-device combinations used in 2019 (n = 2654) and 2020 (n = 519) and longitudinal transitions in cigarette smoking  as a function of ENDS flavor-device combination used in 2019.P = .01), whereas fruit-flavored disposable ENDS use increased from 4.0%  in 2019 to 14.5%  in 2020 (P < .001). Patterns were similar among those who recently quit smoking. Neither cigarette cessation nor relapse rates differed between those who used ENDS that were vs were not prioritized for enforcement efforts .The sample in 2019 included 2654 individuals . Among those who used ENDS and smoked cigarettes, fruit-flavored cartridge ENDS use decreased from 13.9%  in 2019 to 7.9%  in 2020 (In this nationally representative US cohort study of adults who smoked cigarettes and used ENDS, fruit-flavored cartridge ENDS use was nearly halved between 2019 and 2020. Cigarette cessation and relapse rates did not differ between those who used ENDS targeted by CTP and those who used other ENDS. Enforcement Priorities for Electronic Nicotine Delivery Systems (ENDS) and Other Deemed Products on the Market Without Premarket Authorization1 in January 2020. In this report, the FDA prioritized its enforcement efforts against “any flavored, cartridge-based ENDS product (other than a tobacco- or menthol-flavored ENDS product),” which came into effect in February 2020. These priorities were part of a suite of actions intended to reduce ENDS use among young people, which had been rapidly increasing.6 In particular, JUUL, a discreet, pod-style ENDS device that uses replaceable cartridges containing a nicotine salt–based formulation, quickly increased in popularity and became the leading ENDS brand on the US market by 2017.7 JUUL pods were initially available in fruity and sweet flavors, such as fruit medley, mango, vanilla, and crème brulée, although in November 2019, JUUL removed all flavors other than menthol and tobacco flavors from the US market.8The Center for Tobacco Products (CTP) of the US Food and Drug Administration (FDA) published 9 A key concern with ENDS flavor and device restrictions is whether adults who smoke cigarettes will be hindered in quitting smoking and whether adults who have quit cigarette smoking and use ENDS may relapse to smoking if their preferred ENDS products are no longer available.13 However, empirical data on real-world behavior change to support or refute these concerns are limited. A cross-sectional online survey conducted in the US in 2021 (1 year after CTP’s ENDS enforcement priorities became effective) evaluated ENDS flavor use among those who used ENDS in a recent cigarette quit attempt and found no difference in self-report of quitting smoking because of experiencing ENDS flavor restrictions, no difference in quitting because of preferring tobacco or menthol flavor vs another flavor, and no difference in switching ENDS flavors because of experiencing flavor restrictions.14Limiting the availability of fruit- or sweet-flavored ENDS may reduce the appeal of ENDS among youth; however, it may also reduce ENDS appeal among adults because most adults who use ENDS also use non–tobacco-flavored ENDS.15 analyzed data from US adults (aged ≥18 years) who participated in the web-based International Tobacco Control (ITC) Four Country Smoking and Vaping Surveys conducted from February to July 2018 and from February to June 2020, which was during the first few months after the effective date of CTP’s enforcement priorities. The 2020 survey also coincided with the onset of the COVID-19 pandemic in the US, which caused large-scale shutdowns across the nation.16 Gravely et al15 found that more than half of adults who smoked cigarettes and vaped non–tobacco-, non–menthol- (hereafter referred to as fruit-) flavored cartridge ENDS products in 2018 switched to vaping an ENDS flavor-device combination in 2020 that was not prioritized for CTP’s enforcement efforts. In contrast, among adults who smoked cigarettes and vaped an ENDS flavor-device combination other than fruit-flavored cartridge ENDS in 2018, only 6% switched to vaping fruit-flavored cartridge ENDS in 2020. These results are consistent with the hypothesis that CTP’s enforcement priorities may have impacted the vaping behaviors of adults who smoke cigarettes, although the ITC analyses were underpowered to assess potential effects on smoking behaviors, and follow-up data collection was limited to the first 5 months of the enforcement priority implementation in 2020. To address current gaps in the literature, we evaluated adults’ use of ENDS and cigarette smoking after the CTP’s prioritized enforcement efforts against fruit-flavored cartridge ENDS.To date, only 1 nationally representative, longitudinal study has evaluated use of ENDS flavors and device types among adults who used ENDS (at least weekly) and who currently or formerly smoked cigarettes before and after the effective date of the CTP’s enforcement priorities; Gravely et al17 The PATH Study was conducted by Westat and approved by the Westat Institutional Review Board; the study reported here was approved by the Roswell Park Institutional Review Board. All adults provided written informed consent. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.In this cohort study, we analyzed data from adults 21 years or older who participated in the nationally representative, longitudinal Population Assessment of Tobacco and Health (PATH) Study from December 2018 to November 2019  and/or from September 2020 to December 2020 . The 2019 data were collected using in-person, audio, computer-assisted self-interviews. Because of the COVID-19 pandemic, the 2020 data were collected by telephone only.17 Compared with earlier waves, there were differences in the 2020 response rates by socioeconomic status, race and ethnicity, and tobacco use status, similar to other government-sponsored studies conducted during COVID. Importantly, the weighting adjustments were found to essentially eliminate this underrepresentation or overrepresentation, leaving little remaining potential for nonresponse bias in the 2020 estimates.17 Further details on the PATH Study design and methods20 and demographic and tobacco use distributions21 are published elsewhere. Details on interviewing procedures, questionnaires, sampling, weighting, response rates, and accessing the data are available from the National Addiction & HIV Data Archive Program.17The response rates were 88% in 2019 and 56% in 2020, and Nonresponse Bias Analysis Reports have been published for each wave of the PATH Study.17Our analytic sample was composed of adults who used ENDS and currently or formerly smoked cigarettes in 2019 (n = 2654) or in 2020 (n = 519). Longitudinal analyses were among 1013 adults who used ENDS and currently or formerly smoked cigarettes in 2019 and who participated in 2020. Among those who used ENDS and who currently or recently smoked cigarettes, we evaluated differences in the prevalence of ENDS flavor-device combinations used in 2020 compared with 2019. Among those who used ENDS and smoked cigarettes in the past 30 days in 2019, we evaluated smoking cessation in 2020 as a function of ENDS flavor-device combinations used in 2019, and among those who used ENDS and recently smoked cigarettes in 2019, we evaluated smoking relapse as a function of ENDS flavor-device combinations used in 2019. Estimates were weighted to adjust for the PATH Study’s complex study design characteristics  and attrition so that they represent the resident population of the US in 2019 and 2020 who were in the civilian, noninstitutionalized population in 2016 and 2017, as described in detail elsewhere.At each interview, respondents were asked whether they used ENDS  in the past 30 days, and those who had were asked whether they currently use ENDS every day, some days, or not at all. ENDS users were asked, “What flavor is [your regular brand/the brand you last used]? Choose all that apply.” Response options were tobacco-flavored; menthol or mint; clove or spice; fruit; chocolate; an alcoholic drink ; a nonalcoholic drink ; candy, desserts, or other sweets; and some other flavor. We categorized ENDS flavor into the following 4 mutually exclusive categories: (1) only tobacco flavor, (2) only menthol or mint flavor, (3) only nontobacco, nonmenthol, and nonmint flavor(s) , referred to throughout as fruit; and (4) multiple flavors .Those who used ENDS were also asked what kind of ENDS product they use most often . We categorized ENDS device types into the following 3 mutually exclusive categories: (1) disposable product , (2) cartridge product , and (3) tank or mod system ; 1% of those who used ENDS were excluded from analysis because of having an ENDS device type that could not be categorized.We created a 12-level ENDS flavor-device type combination variable as follows: (1) tobacco flavor, disposable; (2) menthol or mint flavor, disposable; (3) fruit flavor, disposable; (4) multiple flavors, disposable; (5) tobacco flavor, cartridge; (6) menthol or mint flavor, cartridge; (7) fruit flavor, cartridge; (8) multiple flavors, cartridge; (9) tobacco flavor, tank or mod; (10) menthol or mint flavor, tank or mod; (11) fruit flavor, tank or mod; and (12) multiple flavors, tank or mod. We also combined these categories into a dichotomous variable to indicate whether the combination was prioritized for CTP enforcement efforts .At each interview, respondents were asked if they smoked cigarettes in the past 12 months and if they smoked cigarettes in the past 30 days. Cigarette smoking status was defined as current smoking if a respondent smoked cigarettes in the past 30 days. Cigarette smoking status was defined as former cigarette smoking if a respondent smoked cigarettes in the past 12 months but did not smoke cigarettes in the past 30 days.Cigarette cessation was defined as no cigarette smoking in the past 30 days in 2020 among those who smoked cigarettes in the past 30 days when interviewed in 2019 . Cigarette relapse was defined as cigarette smoking in the past 30 days in 2020 among those who did not smoke cigarettes in the past 30 days but did smoke cigarettes in the past 12 months when interviewed in 2019 .Respondents also reported their age, biological sex , race , ethnicity (Hispanic or not Hispanic), income , educational level , frequency of cigarette smoking (daily or nondaily), cigarettes smoked per month, menthol vs nonmenthol cigarette smoking, and frequency of ENDS use (daily or nondaily). We included race and ethnicity as covariates in this study because they are known to be correlated with tobacco use. Racial and ethnic groups were combined to protect participant confidentiality.2 tests to determine whether the prevalence of each ENDS flavor-device combination differed between 2019 and 2020, overall and stratified by cigarette smoking status. An α = .05 was considered statistically significant. The 2019 estimates were weighted using the 2019 single-wave weights for the 2016-2017 cohort, and the 2020 estimates were weighted using the 2020 all-waves weights for the 2016-2017 cohort. We estimated variances using the balanced repeated replication method22 with Fay adjustment set to 0.3 to increase estimate stability.23 Data were analyzed from January 1, 2022, to May 2, 2023.We evaluated the cross-sectional prevalence of ENDS flavor-device combination used in 2019 and 2020 among adults who used ENDS in the past 30 days, stratified by cigarette smoking status (current vs former). We used χ22 with Fay adjustment set to 0.3 to increase estimate stability.23We evaluated within-person transitions in cigarette smoking between 2019 and 2020 among adults who used ENDS in the past 30 days in 2019 as a function of whether the ENDS flavor-device combination used in 2019 was prioritized for CTP’s enforcement efforts in 2020 . These analyses were conducted separately among those who smoked cigarettes in 2019  and among those who formerly smoked cigarettes in 2019 . Separate logistic regression analyses evaluating cigarette cessation and cigarette relapse were conducted unadjusted and adjusted for covariates , with α < .05 considered statistically significant. All longitudinal transition analyses were weighted using the 2020 all-waves weights for the 2016-2017 cohort. Variances were estimated using the balanced repeated replication methodThe population in 2019 included 2654 individuals . Of the 2019 population, 53% were aged 21 to 34 years , 35% were aged 35 to 54 years , and 12% were 55 years or older . Of this population, 65% had a household income less than $50 000 , 46% had no college education , 39% had some college or associate’s degree , and 15% had a bachelor’s degree or higher .P = .01), as did the fraction that used menthol- or mint-flavored tank or mod ENDS , whereas the fraction who used fruit-flavored disposable ENDS significantly increased from 4.0%  to 14.5% , as did the fraction who used multiple-flavored cartridge ENDS .P = .11), whereas the fraction who used fruit-flavored disposable ENDS significantly increased from 1.9%  to 14.4% .Among those who used ENDS and who smoked cigarettes in 2019 (n = 876), 25.3% quit cigarette smoking in 2020  . CigaretAmong those who used ENDS and who recently quit smoking cigarettes in 2019 (n = 137), 30.5% returned to smoking cigarettes in 2020  . Cigaret13 Our nationally representative findings show a significant decrease in the prevalence of use of fruit-flavored cartridge ENDS among adults who used ENDS in 2020 compared with 2019, alongside a significant increase in the prevalence of use of fruit-flavored disposable ENDS, with the overall use of fruit-flavored ENDS being similar in each year. Our analysis yielded no evidence of reduced cigarette cessation rates or increased cigarette relapse rates among those whose ENDS products were targeted by CTP’s enforcement priorities compared with those who used ENDS products that were excluded from CTP’s priorities.Concerns have been raised that CTP’s prioritized enforcement efforts against fruit-flavored cartridge ENDS, intended to reduce ENDS use in youth, may negatively impact adults who use ENDS and smoke cigarettes or who recently quit smoking cigarettes.24 It is therefore possible that shifts in adults’ use of ENDS may have contributed to the market share increase in disposable ENDS brands in 2020, such as Puff Bar, which received warning letters from the CTP in July 2020.25This study is the first, to our knowledge, to show an increase in the use of flavored disposable ENDS among adults in the US who currently or recently smoked cigarettes, consistent with the hypothesis that these ENDS users may have switched their device type to maintain their flavor use, which has thus far only been shown among youth in the US.At the same time, our findings show that the potential reach among adults of CTP’s February 2020 enforcement priorities was low. That is, in 2019, only 14% of adults who used ENDS (and who currently or formerly smoked cigarettes) were using fruit-flavored cartridge ENDS, meaning that only approximately 1 in 7 adults were using the specific flavor-device combination that was targeted by the CTP. The low reach and low potential for impact on adults aligns with the intended goal of CTP’s actions to impact youth while considering the whole population when making tobacco regulatory decisions. Although our findings show some ENDS flavor-device product switching among adults and some changes in the national prevalence of ENDS flavor-device combinations used among adults before and after CTP’s enforcement priorities, use of fruit-flavored tank or mod devices remain by far the most common combination of ENDS flavor and device used by adults who smoke cigarettes in the US and the most common combination used by adults who recently quit smoking cigarettes in the US.Although we found no differences in cigarette cessation rates or relapse rates between those who used ENDS that were prioritized for enforcement efforts and those who used other ENDS, we note that sample sizes were small for evaluating transitions in cigarette smoking owing in part to the small segment of adults in the US who were using the targeted ENDS products. Another limitation of this study was including a range of ENDS use and smoking frequencies, and results may differ for those who used ENDS more frequently and/or who may be more dependent on nicotine . We were also unable to distinguish between menthol-flavored ENDS and mint-flavored ENDS because these flavors were combined when participants were queried in 2019. Additionally, 2020 follow-up data were collected within the year after CTP’s enforcement priorities, which was also during the COVID-19 pandemic. Thus, we cannot necessarily attribute observed changes in behavior to the enforcement priorities per se, and analysis of subsequent waves of PATH Study data is needed to evaluate behavior changes in the longer term. Lastly, we evaluated adults 21 years and older, and findings may not be generalizable to those younger than 21 years.The CTP’s February 2020 ENDS enforcement priority to target its enforcement efforts against fruit-flavored cartridge ENDS had low reach among adults who used ENDS and currently or recently smoked cigarettes, consistent with the goal of prioritizing the reduction of youth ENDS use while limiting potential negative effects on other segments of the population. Our findings reveal national-level changes in the ENDS flavor-device combinations used among adults who smoke in the US before and after CTP’s ENDS enforcement priorities, with adults shifting from use of flavored cartridge products to use of flavored disposable products. We found no evidence to support concerns that adults’ cigarette smoking behaviors will be negatively impacted by this effort to protect youth.


Type 1 diabetes is characterized by autoimmune destruction of insulin-secreting pancreatic β-cells leading to disturbed glucose regulation and overt hyperglycemia. Consequently, individuals with type 1 diabetes have a lifelong need for insulin replacement therapy. The primary goal in the long-term care of type 1 diabetes is to maintain glucose levels physiologically as possible, mitigating the risk of micro- and macrovascular complications, which combined represent the primary cause of morbidity and mortality in developed societies . Recent Due to the inhomogeneous nature of physical activity exercise, activity-related hormonal responses, and different gender and age responses to physical activity, a personalized glucose management and nutritional plan should be followed.Daily Challenges Around Physical Exercise, Nutrition and Medication in Type 1 Diabetes covers diverse topics of both pre-clinical and clinical studies, addressing relevant questions surrounding the everyday decisions that individuals with type 1 diabetes encounter to overcome dysglycemia.This Research Topic of McCarthy et al. in their review “The endocrine pancreas during exercise in people with and without type 1 diabetes: Beyond the beta-cell” discussed the crucial role of the endocrine pancreas, beyond its role in digestion and metabolism at rest, in regulating energy during physical exercise. In type 1 diabetes, the destruction of β-cells leads to significant disruptions in glucose control; however, the impact of type 1 diabetes on other pancreatic hormones response during exercise and how they differ from individuals without type 1 diabetes are less investigated. Understanding these responses in both individuals with and without diabetes can help to identify clinically relevant adaptations for effective management of glucose levels around exercise, thus providing optimal clinical care through educated decision making. This knowledge is particularly relevant with the advancement of automated insulin delivery and emerging algorithms that detect physical activity, as it can bridge the gap between clinical and engineering considerations for automatic insulin delivery requirements during physical activity.Barlovic et al. evaluated in their review “Exercise and nutrition in type 1 diabetes: Insights from the FinnDiane cohort” the main findings from the Finnish Diabetic Nephropathy Study (FinnDiane), a long-term research project involving over 8,000 individuals with type 1 diabetes over a period of 25 years. The authors discuss different topics from research in this well-studied cohort, including associations between physical activity, glycemic outcomes, chronic complications, and mortality or associations between different aspects of nutrition, glycemic outcomes, and chronic complications of type 1 diabetes. Importantly, this discussion of the FinnDiane project further underpins the importance of living a physically active lifestyle with type 1 diabetes.Fitzpatrick et al. described in their paper “Exercise, type 1 diabetes mellitus and blood glucose: The implications of exercise timing” that the timing of exercise may play a crucial role in preventing glucose imbalances such as post-exercise hypo- or hyperglycemia. There is limited evidence summarizing the impact of exercise timing on glucose metabolism in type 1 diabetes; in this report, the authors suggest that resistance training or high-intensity interval training (HIIT) should be performed in the afternoon/evening when individuals are more likely to experience hypoglycemia since these types of exercise provide more glucose stability or even an increase in glucose levels. However, continuous aerobic exercise is recommended in the morning due to natural circadian elevations in blood glucose, providing additional protection against exercise-induced hypoglycemia. However, more well-designed studies are needed to further investigate the relationship between exercise timing and glycemic control, ultimately determining the most effective and safe exercise timing for individuals with type 1 diabetes.Rilstone et al. provided in their study “Nutritional support for a person with type 1 diabetes undertaking endurance swimming” information about nutritional considerations for people with type 1 diabetes participating in long-distance open-water events, including personal testimony from a marathon swimmer with type 1 diabetes. Individuals with type 1 diabetes face additional complexities related to insulin management and manage it through high carbohydrate intake. This paper aims to provide insights and recommendations for individuals with type 1 diabetes engaging in long-distance swimming activities, highlighting the main considerations and suggestions for insulin management strategies.Valder and Brinkmann in their opinion article “Is intake of fruit juice useful in exercise-induced hypoglycemia prevention in individuals with type 1 diabetes mellitus?” discussed the role of fruit juice in preventing exercise-induced hypoglycemia in individuals with type 1 diabetes and addressed associated health concerns. The authors provided some practical recommendations regarding nutrition, carbohydrate intake amount, and glucose monitoring before, during, and after physical activity. Very clinically relevant to note for people with type 1 diabetes, when fructose is consumed during exercise, this might further increase systemic lactate concentration, and the extent of blood glucose increase might be lower when compared to glucose consumption.Vlcek et al. reported in their patient-led qualitative study “How we do it: A qualitative study of strategies for adopting an exercise routine while living with type 1 diabetes” the experiences of individuals living with type 1 diabetes on how they adopt and maintain an active lifestyle while managing the risks of hypoglycemia and glucose fluctuations. Semi-structured interviews and focus groups were conducted, and interpretive description analysis was used to identify themes and strategies associated with staying physically active with type 1 diabetes. In their study, the authors found that structure and organization, trial and error learning, psychosocial aspects, diabetes technology, education, and peer support were key facilitators of regular physical activity. Strategies to overcome barriers included utilizing technology, integrating psychosocial support, adjusting insulin and carbohydrate intake, and planning when and how to perform exercise. Their findings emphasize the importance of personalized approaches, understanding individual glycemic responses, and incorporating supportive tools and resources for individuals with type 1 diabetes to maintain an active lifestyle.Gianini et al. demonstrated in their article “Patient reported outcome measures in children and adolescents with type 1 diabetes using advanced hybrid closed loop insulin delivery” that the use of advanced automatic insulin delivery systems resulted in decreased fear of hypoglycemia, less emotional distress, increased quality of life, and reduced burden of type 1 diabetes management together with improved metrics of glycemia. This was investigated by utilizing mixed methods research design using both quantitative and qualitative approaches.Zaharieva et al. in “Adding glycemic and physical activity metrics to a multimodal algorithm-enabled decision-support tool for type 1 diabetes care: Keys to implementation and opportunities” presented an overview of the essential steps of integrating exercise data into an algorithm-enabled patient prioritization and remote patient monitoring program. This care model integrates continuous glucose monitoring data to prioritize patients for weekly reviews by clinical diabetes, has improved clinical workflows, and has been associated with improved glucose outcomes in newly diagnosed young people with type 1 diabetes. Incorporating exercise data (such as step count or heart rate) into the current continuous glucose monitoring (CGM)-based care model could produce additionally clinically relevant information such as identifying whether individuals are meeting physical activity recommendations and would allow for personalized care and better-informed decisions around individual needs, insulin-dosing decisions, and overall diabetes management.Within the last article in our Research Topic, OM: Writing – original draft, Writing – review & editing. KD: Writing – original draft, Writing – review & editing.


The advent of next-generation sequencing (NGS) has allowed for the identification of novel therapeutic targets for patients with uncommon cancers. It is well known that fusion translocations are potent driver of cancer pathogenesis and can render tumors exquisitely sensitive to matching targeted therapies. Here we describe a patient with ALK-fusion positive widely metastatic salivary ductal carcinoma, who achieved a durable complete response from alectinib, a potent and specific ALK tyrosine kinase inhibitor. This case serves as another reminder that ALK-fusions can be targeted regardless of histology and can afford patients dramatic and durable benefit. It also emphasizes the need for insurance coverage for such beneficial therapies. While ALK fusions are exceedingly rare in salivary ductal carcinoma, the presence of multiple other targetable aberrations supports the recommendation for universal NGS testing for such tumors. The treatment of metastatic SDC (mSDC) often includes a platinum-based chemotherapy regimen (i.e. doxorubicin and cisplatin) with modest benefit2. The 5‐year and 10‐year survival rates for mSDC are 43.5% and 14.5%, respectively3. It is widely known that mSDC can express androgen receptor (AR) and harbor HER2 amplifications mimicking features of prostate and breast cancer. Hence both AR and HER2 staining by immunohistochemistry is often considered. The responses to HER2 based therapies have been reported at 60–70% and to AR blocking agents between 18 and 53%2. Furthermore PD-L1 expression is variable, but durable benefit has been described with immune checkpoint inhibition4.Salivary ductal carcinoma (SDC) is a rare and aggressive cancer found mostly in the parotid salivary gland in older patients. It is associated with a poor prognosis, often characterized by rapid progression, early metastatic spread, and a high rate of disease recurrence after surgery and adjuvant therapy5. This coupled with the continued rapid development of targeted therapeutics can help to get the right drugs to the right patient even for rare cancers8. Fusion events in particular are oncogenic drivers of carcinogenesis and ideal targets with dramatic responses9.Next-generation sequencing (NGS) has allowed for a more comprehensive identification of novel therapeutic targets which can lead to improved outcomes for patients with such oncogene-driven cancers11. Despite this finding, there have not been any reports of using an ALK-directed therapeutic strategy to treat these patients.There has been a paucity of case reports that describe various ALK rearrangements in patients with mSDC. Various studies have found SDC to harbor ALK rearrangements in 4–6% of cases; a rare event in a rare tumor14. The n-of-1 approach to customize therapies based on patients’ NGS reports is likely paramount to improving outcomes in metastatic cancers15. This case represents employment of such an approach with a favorable outcome for a patient with mSDC.Tissue agnostic approvals have allowed for patients to receive therapies regardless of the histology of origin and have led to significant clinical benefit. Targeting ALK-fusions in other histologies aside from non-small cell lung cancer has been shown to derive both radiographic response and clinical benefitA 65-year-old woman initially noted swelling of the right parotid. Initial workup included cross-sectional imaging with a computed tomography (CT) scan which did not reveal a discrete lesion; however, noted thickening of the parotid tail. Swelling progressively worsened and 4 months later she underwent an ultrasound-guided biopsy, which revealed a 2 cm mass. Pathology was reported as a salivary gland neoplasm of uncertain malignant potential and again observation was chosen. However, the neck mass continued to grow, and a follow up neck CT was notable for an infiltrative lesion in the right parotid gland measuring 4.2 × 1.7 cm with possible metastatic nodes in the adjacent right neck measuring up to 12 mm. The patient underwent a right parotidectomy with ipsilateral neck dissection. The surgical pathology revealed a high-grade, poorly differentiated carcinoma diffusely infiltrating the salivary gland with extra-glandular soft tissue extension and perineural invasion. There were 24 positive lymph nodes in the 37 examined with the largest lymph node measuring 2 cm with extra-nodal extension. Immunohistochemical stains demonstrated that tumor cells were positive for CK7, GATA3, and androgen receptors, but negative for HER2, CD117, ER, PR, p53, p63, and SOX10 with a Ki-67 index of approximately 50%. She underwent a positron emission tomography (PET) scan after surgery, which showed an additional hypermetabolic right level 1B lymph node consistent with metastasis, and numerous hypermetabolic lesions in both hepatic lobes consistent with both locoregional and distant metastases. Of note numerous osteolytic and CT occult hypermetabolic lesions were noted and consistent with widespread osseous metastases including involving bilateral ribs, cervical, thoracic, lumbar, and sacral vertebra, bilateral pelvis, and left femoral neck Fig. . A CLIA-The patient was started on alectinib 600 mg twice daily for which she paid herself due to denial by the insurance company despite multiple appeals. The patient had severe osseous pain spending the majority of her days in bed at the time of initiation of alectinib.Within two weeks of starting alectinib she had marked improvement in symptoms. A PET scan performed after one month of alectinib therapy compared to pre-treatment showed a complete resolution of the hypermetabolic lymph node at right level 1B previously seen and complete resolution of FDG avid focus within the right level 2A, complete resolution of abnormal metabolism of all multiple hypermetabolic liver nodules, no new hepatic hypermetabolic lesions with FDG above liver background . In this case as seen in NSCLC the superiority of alectinib compared to an earlier generation ALK inhibitor, such as crizotinib, the authors wanted to avoid potential resistance that can occur with crizotinib, but that alectinib can overcome as well as the lower rates of toxicity. Without an attempt to target such alterations, it is hard to predict response and furthermore may render a tumor resistant to other therapies20. Given the rarity of ALK translocations in salivary duct cancers there will not be any trial or FDA approval short of a tumor agnostic approval, which the FDA has recently approved for other oncogenic aberrations 21. The approval and insurance coverage process is problematic for rare tumor types with rare targetable alterations, and such patient undoubtedly oftentimes fare poorly in our current health care system22. Despite this, broad genomic profiling using a large panel for patients with mSDC may provide insight into novel therapeutics as it did for our patient.This brings up the question of ‘right to try’ for patients with metastatic cancers as well as the importance of compassionate use programs from pharmaceutical companies, which often help26. The authors report a case of a patient with ALK-fusion mSDC receiving alectinib and achieving a complete response; additional studies are warranted in this patient population.Our patient continues to have clinical benefit with an ongoing complete response eight months after therapy initiation, which highlights the importance of precision medicine and routine molecular profiling in such cases to address some of the inequities apparent for rare tumorshttps://strataoncology.com/), a CLIA-certified laboratory, where NGS and RNA sequencing (https://strataoncology.com/wp-content/uploads/2022/12/Gene_List_SO-SPEC-003v7.pdf) was performed on the resected specimen, which identified an EML4-ALK fusion. The patient was started on an ALK inhibitor, alectinib, and had complete resolution of her cancer which is ongoing at eight months.A 65-year-old woman underwent a CT for facial swelling with a biopsy that confirmed SDC with immunohistochemical stains which were positive for CK7, GATA3, and androgen receptor with a Ki67 index of approximately 50%, and negative for SOX10, p63, CD117, estrogen receptor, progesterone receptor, and p53. She also had a PET-CT scan that showed metastatic disease and her tissue was sent to Strata Oncology (Further information on research design is available in the REPORTING SUMMARY


The refractory metal–oxide semiconductors arean overlookedplatform for nanophononics that offer alloys with high melting pointsand tunable optical constants through stoichiometry changes and ionintercalation. We show that these semiconductors can form metamaterialcoatings (metacoatings) made from a set of highly subwavelength, periodicmetal–oxide layers (≤20 nm) with a varying and gradedrefractive index profile that includes a combination of high and lowrefractive indices and plasmonic layers. These metacoatings exhibitvibrant, structural color arising from the periodic index profilethat can be tuned across the visible spectrum, over ultralarge lateralareas through bottom-up thermal annealing techniques. Notably, alloys such as WO3, Fe2O3, CuO, Cu2O, and CeO2, in particular have higher dielectric resonator figures ofmerit than those offered by popular dielectrics widely used in nanophotonicssuch as GST, BST, VO2, GaP, GZO, and ITO across the visiblewavelength range.5The unprecedented functionalitiesand electromagnetic properties engineered using the metamaterialsand metasurface paradigm in various nanophotonic device platformsthroughout the past decade arise from subwavelength periodic and/ornonperiodic metamolecule resonators embedded within a host dielectricor plasmonic medium of a particular chemical composition. Precisemicro-/nanostructuring of the geometry of the resonators enables therealization of remarkable electromagnetic behavior due to the engineeredeffective permittivity and permeability, and by extension the refractiveindex. Photonic metamaterials research has traditionally focused onnoble plasmonic metals which afford no compositional tunability, relativelylow-melting-points, high optical losses, and a lack of CMOS compatibility.To combat these losses and instabilities, researchers are activelyexploring alternative material platforms to plasmonic metals suchas chalcogenide semiconductors, transparent conductive oxides, nitrides,and 2D materials.7 In both cases, thermal oxidation startswith the impingement of oxygen molecules (O2) onto a heatedmetallic film. The molecular oxygen is physiosorbed onto the surfaceand dissociated into oxygen (O) atoms that are then chemically bondedto metal atoms (M) at the film’s surface.8 Thebonded oxygen and metal atoms reorientate their chemical bond intothe lowest energy state by exchanging the internal M atom with theexternal O atom driving the O atom into the metal film. This process,known as “place exchange”, leads to the growth of theoxide film one monolayer at a time. The thermal oxidation proceedsby the transportation of oxygen and metal ions through the metal–oxidemonolayers to the metal or air interface, where additional oxidationreactions can occur, leading to an increase in oxide film thicknesswith time.11 Therefore, as film thickness increases the additionaloxide volume does not strictly form with the same concentration ofO or M atoms across the thickness of the film. Depending on the Mand O species used, this can result in the formation of multiple discreteoxide layers with graded stoichiometric profiles, due to the nonlineardiffusion profiles of M and O ions across the thickness of the film.11 The resulting concentration gradientsformed across the thickness can lead to the formation of films withgraded refractive index profiles that can be precisely tuned throughthe thermal oxidation process. The oxidation process, if left to continue,eventually homogenizes all monolayers through the oxide film untilthe metallic film is fully oxidized, the film temperature drops belowthe oxidation temperature of the metal, or the oxygen gas is removedor depleted from the oxidation environment. Therefore, during thethermal oxidation of metals, below a homogenization threshold, thevarying diffusion of metal, metal oxide, and oxygen species can leadto a stoichiometric grading across the thickness of the oxide whichtranslates to graded optical properties across the nanoscale thicknessof the grown metal–oxide film.15 The resulting optical propertiesarise from the interplay of the graded refractive index profile ofall the layers and can act as the basis for a myriad of optoelectronicfunctionalities.Refractory metal–oxidescan be directly deposited throughphysical vapor deposition techniques or grown from a seed metalliclayer by oxidation. The oxidation of metals is a thermal process thatcan occur through direct annealing or laser-induced oxidation of metallicfilms.x coating in resistive RAM(RRAM) technologies to achieve high endurance and thermal stabilityin nonvolatile memory devices.16 AdditionallyHfxAl1–xOy films with a graded Al profile have been shown toexhibit multilevel electronic switching capabilities that can be usedto form an artificial synapse for neuromorphic computing applications.17 Most notably, graded transparent conductiveoxide films have also been explored as antireflective optical coatingsor GRIN materials based on graded cosputtering of ITO or TiO2 with SiO2 to form multilayer stacks.19 Though the exploration of graded films is typically emulated bymodulated deposition routines few researchers have found thermal annealingof parent materials  can be performed to form SnO2, In2O3, and ITO which exhibit an opticalresponse that can best be described when employing graded opticalconstants.21 Furthermore, films with controlled,graded optical constant have been shown to hold great promise foreven wider nanophotonic applications and can be used as graded indexlenses in super-resolution microscopy, and can provide better resolutionin photolithography techniques.24Thin films having graded elemental contenthave found use in datastorage, protective coatings, and as optical materials. Electricalcontacts in the form of W plugs can be thermally annealed to forma graded WOx) coating with subwavelength overall thickness, and highlysubwavelength interlayers that form a large lateral area color-tunablecoating on a seed tungsten (W) surface. In our experiments, W filmsare deposited through sputtering and used as seed layers for thermaloxidation at elevated temperatures (up to >400°C) in ambientatmospheres (methods). Post thermal oxidation,below the homogenization threshold, a mixed phase tungsten oxide filmis obtained as observed through X-ray diffraction (XRD) measurements, we demonstratethe bottom-up growth of a graded tungsten oxide  is shown in n > 3) upper layers close to the metal–oxide air interfaceand low-index, plasmonic buried layers that are comparable to vacuum(n ≥ 1) at the metal—metal–oxideinterface .The number of layers that best describes the optical performance ofthe metacoatings increases with overall thickness showing an increasefrom four to seven, of approximately L = 20 nm thicklayers, when increasing the overall film thickness from T = 83 to 134 nm.The dispersion of refractiveindex and extinction coefficient ofthe individual layers for the case of a Figure S1 for the simulated angle dependenceof the response).To further confirm this, finite-difference time-domain(FDTD) simulations4d,e, are3 film through the reactiveRF sputtering PVD technique. Variable angle spectroscopic ellipsometrymeasurements and subsequent FDTD simulations reveal that, in contrastto the thermally oxidized metacoatings, as this film has been grownthrough a homogeneous vapor deposition method, it does not possessand rely upon a multilayered graded index profile as the basis forits observed optical response. Here, a single layer best describesthe observed response . Furthermore,metal–oxides are a widely used platform in electronic memristivetechnologies where movement of oxygen atoms within a host latticeprovides the means for changes in conductivity and refractive indexthat can be exploited in subsequent reconfigurable metacoating deviceplatforms. Therefore, their use within nanophotonic metamaterial andmetacoatings will open the door to reconfigurable and adaptive devicesenabled by the intercalation and movement of metal and gas ions withinthe metal–oxides.30 Metal–oxidematerials are also sensitive to the chemistry of their surroundingenvironment and have been widely used in environmental monitoringand optoelectronic gas sensing platforms, which can be exploited infuture flat optical sensing devices using metacoating technology.5In particular, the metal–oxidemetacoatings presented inthis work can be grown on a variety of surfaces from wafer-scale tolarge industrial-scale parts and components, using localized laseroxidation or by large area thermal oxidation through the use of high-capacityfurnaces. Based on the chosen oxidation temperature, tailored oxidationtimes can be achieved (


Pathogen infection resulted in a distinct microbial community structure and functions on the susceptible cultivar, while concurrent O3 stress did not further alter the community structure, and function. However, O3 stress exacerbated the disease severity on resistant cultivar. This altered diseased severity was accompanied by enhanced heterogeneity in associated Xanthomonas population counts, although no significant shift in overall microbiota density, microbial community structure, and function was evident. Microbial co-occurrence networks under simultaneous O3 stress and pathogen challenge indicated a shift in the most influential taxa and a less connected network, which may reflect the altered stability of interactions among community members. Increased disease severity on resistant cultivar may be explained by such altered microbial co-occurrence network, indicating the altered microbiome-associated prophylactic shield against pathogens under elevated O3. Our findings demonstrate that microbial communities respond distinctly to individual and simultaneous stressors, in this case, O3 stress and pathogen infection, and can play a significant role in predicting how plant-pathogen interactions would change in the face of climate change.While the physiological and transcriptional response of the host to biotic and abiotic stresses have been intensely studied, little is known about the resilience of associated microbiomes and their contribution towards tolerance or response to these stresses. We evaluated the impact of elevated tropospheric ozone (O The phyllosphere (aboveground parts) of plants is a unique, nutrient-poor habitat and is inhabited by various prokaryotic and eukaryotic microorganisms  that col2  in the control chambers was around 30.6 ppb, while the fumigated chambers had an average [O3] of about 90.3 ppb  while the O3 levels between the elevated chambers were similar (p = 0.62).The experiment was conducted at the Atmospheric Deposition (AtDep) site at Auburn University Fig.  in the 2 O3 Fig. . Each elber Fig. . The plappb Fig. . O3 leveX. perforans suspension adjusted to 106 CFU/ml in MgSO4 buffer amended with 0.0045% (vol/vol) Silwet L-77 . The control plants were dip-inoculated in MgSO4 buffer amended with 0.0045% (vol/vol) Silwet L-77  were inoculated with the pathogen X. perforans while 6 chambers (three ambient and three elevated O3) had control plants inoculated with MgSO4 buffer , followed by two other time points during the growing season (mid and end of the season). For each timepoint, leaves from 6 plants of each cultivar grown inside one chamber were pooled, so we have one sample per cultivar. During sampling, leaves were collected randomly to avoid bias towards diseased leaves and with at least one leaf per plant for each cultivar. 40 grams of leaf samples were sonicated for 15 min in phosphate-buffered saline solution (50 mM) amended with 0.02% Tween 20 and the dislodged cells were pelleted down and processed for DNA extraction. Briefly, total DNA was extracted using Wizard® Genomic DNA Purification Kit  as per manufacturer instructions with the addition of a phenol:chloroform:isoamyl alcohol (25:24:1) followed by ethanol precipitation. The DNA was quantified using a Qubit 3.0 fluorometer  and the DNA samples were submitted to the Duke Center for Genomic and Computational Biology sequencing core  for library preparation, and paired-end reads (2 × 150 bp) were sequenced on NovaSeq 6000 S1 flow cell system. The raw reads were then trimmed for quality using BBDuk (http://jgi.doe.gov/data-and-tools/bb-tools/) followed by host contamination removal with KneadData (https://bitbucket.org/biobakery/kneaddata/) using pepper cv. 59 (GCA_021292125.1) genome as a reference.Pepper leaf samples were collected from both inoculated and control samples of each cultivar separately after inoculation with https://github.com/smdabdoub/kraken-biom) was used to convert the output from Bracken into BIOM format tables for diversity analyses in R  can increase exposure to pathogens, thus, impacting disease severity [3. Secondarily, increased pathogen virulence via increased effector output [Studies on plant’s response to a combination of abiotic and biotic stress have shown a unique and more complex response than that of individual stresses , 91–93. severity . Complemr output  under al3 and pathogen infection. Microbial communities recruited by the resistant cultivar in the phyllosphere could have a protective role against the pathogen as it has been demonstrated in previous studies [3, which may have led to increased disease severity. The bacterial and eukaryotic community composition, structure and function on the susceptible cultivar did not differ in the absence of pathogen infection or elevated O3. However, bacterial community structure on the resistant cultivar were influenced by presence of elevated O3, but in absence of the pathogen. Whether such differential influence on microbial community structure is due to specific resistance loci remains to be determined since the cultivars that we investigated were not near-isogenic lines for the resistance loci. On the susceptible cultivar, the presence of pathogen infection caused a sizeable shift in the bacterial community structure and function, even though concurrent O3 stress did not further alter the microbiome structure and function. Although no significant shift in microbiome structure and function was observed on the resistant cultivar upon infection, overall microbiota density associated with infected resistant cultivar was lower compared to infected susceptible cultivar. Furthermore, concurrent O3 stress resulted in lower total microbiota density during mid-season sampling on the infected resistant cultivar. Whether such reduction reflects impaired prophylactic potential of microbiome associated with resistant cultivar under the combined impact of elevated O3 and pathogen infection remains to be investigated. Further experiments to assess the microbiome-mediated protection against pathogen can be designed using synthetic communities associated with the resistant cultivar, similar to the previous studies [Third and the most important explanation for the observations from this study is the alteration in microbiome-mediated protection on the resistant cultivar in response to elevated O studies , 100 and studies . These e studies –104.3 stress and simultaneous pathogen and O3 stress showed a similar trend of a relatively unstable random network compared to the control environment. This finding agrees with the notion that varying degrees of environmental stress disturb the stability of microbial communities [3 stress considerably affected hub taxa. However, simultaneous stressors, but not individual stresses, had considerable influence on the most influential taxa, suggesting that plants respond to simultaneous stresses by changing the most influential microbial member in the random network. It would be interesting to dissect further the influence of individual cultivar and, thus influence of host defense responses on microbial community networks, as we observed a strong cultivar effect on community composition. However, the present study is limited in sample size, which does not allow sufficient power to compare the network structure across individual cultivars. As we observed that elevated O3 impacted eukaryotic communities more strongly than bacterial communities and pathogen infection impacted bacterial communities, influence on cross-kingdom interactions cannot be ruled out in this case. Nevertheless, the present study has limitations in determining how specific and concurrent stressors affect cross-kingdom interactions due to the absence of appropriate methods to evaluate relative abundance of eukaryotic communities using shotgun metagenome data. It is possible that elevated O3 will have an impact on cross-kingdom interactions, as has been shown with other abiotic stressors [Microbial function in the ecosystem is determined not just because of the number and composition of taxa but also the various positive, negative, direct, or indirect associations among the community members . In respmunities . The obstressors .Overall, our study demonstrated that microbial communities respond to a change by not only altering community composition but also interactions among members and overall community function. This work provides a base for our understanding of the complex response of microbial communities and their interactions with the host genotype in response to a changing climate. As plants have evolved in association with their phyllosphere microbiome members, the community members identified in this study have shown to be particularly susceptible to a shift in response to abiotic stress or combined stress. Findings from this study are crucial to evaluate for future work on harnessing the microbiome for stress-tolerant plants.Supplementary materials