Virtually all of these reviews have focused on quantitative approaches aiming at developing an understanding of the underlying mechanisms behind photocatalytic behavior and the parameters that influence structure–function correlation. Less attention has been paid, however, to qualitative measures around the development and assessment of photocatalysts. These measures will contribute toward narrowing the range of potential photocatalytic materials for widespread applications. The current report provides a critical perspective over some of the main factors affecting the assessment of photocatalytic materials as a code of good practice. A case of study is also provided, where this qualitative analysis is applied to one of the most prolific materials of the last-decade, disorder-engineered, black titanium dioxide (TiO So far, these efforts have rightly focused on the interplay between material properties and photocatalytic activity; i.e., they account for a quantitative description of the main parameters that potentially contribute to their photocatalytic performance  the preparation method and experimental factors affecting key material properties toward photocatalytic performance, (2) characterization approaches and synergy between theory and experimental work, (3) photocatalytic tests and control experiments, and (4) material stability within a broad range of experimental conditions. Attention to these qualitative approaches is widely implied but often disregarded in the literature. Drawn together over the edges of a pyramidal diagram (2–widely used as self-cleaning products in the market—and zinc oxide (ZnO). In contrast, promising materials, such as those based on perovskite structures, which may show outstanding efficiencies but only under restricted conditions  nanorods  nanorods  using chnanorods . The aut = 5 min . Analysi2–used as a case of study below—showed different optical properties, either black or blue products, from otherwise similar synthesis conditions using either stainless steel or quartz reactors, respectively  and following different synthesis approaches. It can also challenge the reproducibility of the chosen approach and provide information about its potential scalability. Following a single procedure, the potential influence of reactor elements must be carefully considered. A study on the synthesis of disorder-engineered, hydrogenated TiO2 films under relatively low-temperature conditions over a rutile substrate  films deposited on tin-oxide-based supports  lattice. But perhaps more relevant to experts in material engineering is to consider the expected translation of functional properties upon scaling up from the nano- to the macroscale. Discussion on some key size-dependent phenomena in photocatalysis, such as charge transport, has been presented previously  under continuous supercritical-water conditions  interactions, which may induce charging or coupling of quantum states, for example in novel plasmonic photocatalysts.Issues related to an inadequate or incomplete characterization of nanomaterials have been widely discussed ; chemical stability (no changes in oxidation states of surface species or in the concentration of oxygen vacancies during reaction conditions); and surface inertness . Instead, a more accurate interpretation of the action painting of a semiconductor can be established using in situ techniques with in-depth material characterization during irradiation, i.e., while the photocatalyst is in an excited state, and ideally under reaction (in operando) conditions. This approach is widely established in thermal catalysis  X-ray spectroscopy techniques, with emphasis into photoelectrochemical water splitting and hydrogen evolution processes  is the most common core technique for phase identification and structural information of materials. Several issues have been raised with regards to the limitations of XRD analysis and the interpretation of XRD patterns Ohtani, . The concrystallite size using the Scherrer equation with little attention to its associated restrictions. The Scherrer equation (dhkl = Kλ/βhklcosθ) allows for an estimation of the coherent diffraction domain size in the direction perpendicular to the lattice planes with Miller indices hkl, using a numerical  factor K and the full-width at half-maximum (βhkl) and Bragg angle (θ) of the corresponding diffraction peak for a given wavelength (λ). Since the analysis is sensitive to a particular direction, special care must be paid in the case of highly anisotropic crystallite morphologies, such as needles or disks  and atomic force microscopy (AFM). The main advantages and limitations of these techniques have been extensively reviewed  is a powerful and versatile lab-based core tool for the analysis of surface composition. This technique provides qualitative and quantitative chemical information from the near surface region of films and particles, and thus, it is of fundamental importance in the characterization of catalytic materials Venezia, . The limThe optical properties of photocatalytic materials is widely studied using spectrophotometry. The optical bandgap is typically estimated using the Tauc method from absorbance analysis Murphy, . The connot in contact—can be compared. A more relevant picture from an experimental viewpoint would include the equilibration of the fermi level over the interface and, particularly, the fate of this fermi level in equilibrium into quasi-fermi levels under illumination. This information is, however, out of reach for DFT calculations. Further insight in this sense may be sought from complementary computer-aided design methods together with advanced characterization techniques, such as transient absorption spectroscopy  or any sacrificial agents (reagent blanks) used in the experiments. It is also important to gather information of the process in the dark in order to rule out potential reactions driven by chemically active surfaces. Whenever possible, it will also be useful to study the behavior of visible light-active photocatalysts irradiated under wavelengths outside the material absorption range, as discussed for action spectra analysis below.Control experiments are key toward establishing the nature and appropriate quantification of a photocatalytic process. Yet, it is still surprising to find broad claims of enhanced photocatalytic activity without attention to adequate control tests. These include processes in the absence of substrate or photocatalyst   or a broader range of conditions and how these would affect the material properties (Method Dependence). Unfortunately, minor modifications in the synthesis procedure seem to have a critical impact in its physical and functional properties, as discussed below. The potential scalability of this material has not been addressed .The engineering of TiOmaterial , designematerial . It was Material Properties and Characterization) has been used to study black TiO2 including in situ advanced techniques (In Situ/Operando Techniques: Structure–Function Correlation) such as X-ray absorption/emission spectroscopy  as well as core techniques such as X-ray diffraction, Raman spectroscopy, and nuclear magnetic resonance . For almost all hydrogenation approaches, disordered structural features were observed near the surface of black TiO2 with a general lattice contraction of the disordered layer, although expansion of the shell has also been reported . Most of these features have not been observed in subsequent work, and emerging bands and band shifting in some cases have been attributed to other factors, such as phonon confinement, the presence of oxygen vacancies and other TiO2 phases, among others , scanning transmission electron microscopy (STEM), X-ray absorption fine structure (XAFS), and X-ray absorption near edge structure (XANES) , although not all hydrogenation procedures result in black TiO2  indicated that the lattice disorder in black TiO2 would come from the interplay between surface Ti–O bonding breaking upon adsorption of hydrogen, with formation of Ti–H and O–H bonds, and the desorption of hydrogen molecules in the bulk . At this point, it is worth noting that broad light absorption does not necessarily correlate with extended photocatalytic activity. The initial study  as a hydrogen cocatalyst and methanol as a sacrificial agent under UV-light irradiation. The energy conversion efficiency (defined as the ratio between the energy of solar-produced hydrogen and the energy of the incident sunlight) was 24% for black TiO2, which significantly exceeded that of typical TiO2-based materials (ca. 2%). The enhanced photocurrent in the UV range was attributed to an increase in donor density due to oxygen vacancies formed upon hydrogenation of TiO2. The high electronic density will contribute to shift the Fermi level of TiO2 toward the conduction band, enhancing charge separation and transport with an optimum concentration of oxygen vacancies. The authors used the original sample before hydrogenation as a blank sample (Control Experiments and Blank Samples); other blank samples, such as those obtained under similar annealing posttreatment under air or inert (argon-flow) atmosphere, would have been appropriate.The photocatalytic performance of black TiODye-Sensitized Processes). Among the few examples excluding dye-sensitized processes . For instance, in one case, the TiO2 nanotubes collapsed into white TiO2 nanorods after the relevant annealing treatment . The drop in photocatalytic activity of black TiO2 under visible-light irradiation has been widely observed in the literature  showed a decrease in activity from 95% in the UV range (370 nm) to ca. 1% in the visible range (>420 nm)  confirmed that the sample was not merely a reservoir of hydrogen, and the amounts of hydrogen detected significantly exceeded that contained in the sample after hydrogenation. To the best of our knowledge, no further details have been provided about the long-term stability and performance of this material. The structure–property landscape of black TiO2 remains largely incomplete, and it will undoubtedly be subject of further active research throughout this new decade. Yet, following the photocatalytic work reported so far, this promising material will remain at the bottom of the qualitative pyramid shown in Cycling experiments  (Materiaoperando conditions and from closer synergy between theoretical and experimental studies (Tong et al., The area of photocatalysis may have reached maturity from a fundamental perspective Ohtani, , but theDespite fruitful research in photocatalytic materials (Tong et al., The article was developed and written by RQ-C with scientific input and further discussion by IP. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The exercise intervention alone improved several dimensions of physical fitness  and cognitive function . Relative to exercise training alone, the multimodal fitness and nutritional intervention further improved working memory (+ 9.0%), fluid intelligence reaction time (− 7.7%), processing efficiency (+ 1.8%), heart rate (− 2.4%) and lean muscle mass (+ 1.5%). These findings establish the efficacy of a multimodal intervention that incorporates aerobic fitness and strength training with a novel nutritional supplement to enhance military performance objectives and to provide optimal exercise training and nutritional support for the modern warfighter.Achieving military mission objectives requires high levels of performance from Airmen who operate under extreme physical and cognitive demands. Thus, there is a critical need to establish scientific interventions to enhance physical fitness and cognitive performance—promoting the resilience of Airmen and aiding in mission success. We therefore conducted a comprehensive, 12-week randomized controlled trial in active-duty Air Force Airmen ( A primary aim of research in the nutritional sciences is therefore to establish and validate nutritional interventions for the modern warfighter that are designed to provide optimal nutritional support and supplementation for body and mind. There is growing interest within the United States military to understand the effects of nutritional factors on warfighter resilience examined with respect to both physical and cognitive performance3.Adaptive military operations are a hallmark of modern warfare and depend on the capacity for flexible, resilient behavior. Airmen face extreme physical and mental demands that require the capacity to overcome the negative physical and psychological effects of operating in challenging field environments characterized by “volatile, uncertain, complex, and ambiguous” events4. Acute stress in special operation soldiers may impair visuo-spatial capacity and working memory due to high dopamine and norepinephrine turnover in the prefrontal cortex, which is known to impair cognition and spatial working memory5. Surveys among military populations indicate that most warfighters use multivitamins, protein supplements or sports bars/drinks with a perception that these supplements improve their performance7. However, the efficacy of many of these supplements is not supported by randomized controlled trials9. Caffeine benefits executive function in sleep-deprived individuals; but the effects are acute and have negative side effects in some individuals10. Recent evidence further suggests that some supplements may contain dangerous or illegal substances that can have unknown consequences11. Thus, an enduring aim of research in the psychological and brain sciences is to establish nutritional supplements to enhance physical fitness and cognitive performance in active-duty military populations.Challenging field environments can prevent warfighters from realizing their optimal physical and cognitive performance. Participants with higher levels of stress and anxiety in military survival training exercises have lower scores on the Army Physical Readiness Test16. Elements of the Mediterranean diet are known to promote healthy vasculature by way of reducing total cholesterol and low-density lipoproteins 17, and improving endothelial function18. Broadly, these actions lower the risk of vascular comorbidities, dyslipidemia, hypertension, and coronary artery disease19. Within the brain, these actions are linked to a reduction in white matter lesions and promotion of white matter microstructure19. Furthermore, improvements in white matter brain structure are known to enhance cognitive performance . Thus, combinations of these nutrients in the form of a novel nutritional supplement have the potential to improve the protective vascular, metabolic, antioxidant, and anti-inflammatory mechanisms promoted by individual nutrients and therefore to enhance brain function and cognition13.Accumulating evidence suggests that nutrients found in the Mediterranean diet promote brain and cognitive health. The Mediterranean diet includes olive oil as a source of monounsaturated fatty acids and polyphenols, fish and salmon that deliver phospholipids, omega-3 polyunsaturated fatty acids, lutein and vitamin D, and fruits and vegetables that provide antioxidants and vitamins B, C and E, carotenoids including lutein, folate, and polyphenols21. Specifically, these models have demonstrated that exercise interventions result in (1) hippocampal neurogenesis; (2) increased synaptic activity in the brain; (3) growth of new blood vessels; (4) increased concentrations of brain derived neurotrophic factor (BDNF); (5) reduction of neurodegeneration; and (6) enhanced learning and memory21. The results from these animal models provide the basis for human studies of physical activity, fitness, and exercise. A meta-analysis of exercise interventions in humans revealed a moderate effect size for exercise leading to better cognition23. The meta-analysis showed that exercise improved a variety of cognitive tasks; but the largest effects were observed in tasks that engage the central executive network, including planning, problem solving, and working memory. Finally, the meta-analysis demonstrated that the combined effects of aerobic exercise, power and flexibility training had a greater cognitive benefit compared to only aerobic exercise training.Physical activity interventions improve cognition and the brain in multiple animal models, including rodents, dogs and monkeysMotivated by these parallel lines of research, the present study examined the efficacy of a multi-modal fitness and nutritional intervention to enhance cognitive performance and physical fitness in United States Air Force Airmen. Specifically, we investigated: (1) whether a unimodal exercise training protocol enhanced fitness and cognition and (2) whether a multimodal fitness plus nutritional intervention resulted in fitness and cognitive gains beyond those conferred by the unimodal intervention.Study participants were active-duty Air Force Airmen recruited from Wright Patterson Air Force Base in Dayton, Ohio from January of 2016 through May of 2018. Study eligibility required participants to: (1) have active-duty status; (2) commit to study participation for 14 consecutive weeks; (3) be at least 18 but no older than 45 to minimize the risk of physical injury or cardiovascular occurrence due to the study’s required fitness activity; (4) not have a Department of Defense medical profile for mental and/or physical function limitation nor have a pregnancy profile; (5) not currently be taking prescription blood pressure medication; (6) cease taking herbal dietary supplements, performance supplements or any other substance that contained ingredients that might affect cardiovascular response with exercise one week before study participation began; (7) not have musculoskeletal injury that would limit their ability to engage in heavy resistance training and aerobic exercise; (8) not have cardiovascular or respiratory disease that would limit their ability to engage in heavy resistance training and aerobic exercise. 217 participants enrolled in the study; 205 began the intervention; and 148 completed the study. Only the data for those who completed the study were analyzed in this study and their demographics are presented in Table 24. Informed consent was obtained from all participants in this study. All methods were carried out in accordance with Air Force Research Lab regulations. Participants were recruited at Wright Patterson Air Force Base through IRB approved flyers and emails.All experimental protocols were approved by the 711th Human Performance Wing Institutional Review Board (IRB). The 711th Human Performance Wing is part of the United States Air Force and comprises three units: the Airman Systems Directorate, US Air Force School of Aerospace Medicine and the Human Systems Integration DirectorateParticipants were randomly assigned to either the exercise plus nutritional supplement group (n = 70) or the exercise plus placebo group (n = 78). The sample size was based on a two-arm power analysis that specified an effect size of 0.20 and power of 0.90 and an attrition rate of 20%. Intervention assignment was counterbalanced for the 7 cohorts (about 25 participants per cohort) such that odd numbered cohorts received the supplement and even numbered cohorts received the placebo. Both experimenters and participants were blinded to group assignment. The exercise training intervention was identical for both groups. A battery of cognitive and physical fitness assessments was administered at pre- and post-intervention. This clinical trial was registered with the ISRCTN registry, number ISRCTN96342002, and was approved on July 28, 2020. This clinical trial was assigned a United States Air Force clearance of CLEARED on 28 Jul 2020 with case number MSC/PA-2020-0172.The exercise intervention was designed to enhance mission relevant strength, cardiovascular health and fitness. The training consisted of a total body resistance circuit designed to improve upper- and lower-body strength (Monday and Wednesday); a moderate cardiovascular workout designed to facilitate active recovery, flexibility and core strength (Tuesday and Thursday); and a cardiovascular endurance training and high intensity cardiovascular workout (Friday). Fitness training sessions were scheduled five days a week (Monday–Friday) for 12 weeks and session each lasted 45 minutes. Participants were guided on proper form for each exercise. Air Force Research Lab research staff, consisting of exercise physiologists, athletic trainers and strength coaches, designed and supervised the exercise intervention. All exercise sessions were supervised by at least one member of the research staff. Average attendance rate for the fitness training sessions was 90%. Supplementary Fig. https://creativecommons.org/licenses/by-nc/4.0/.The nutritional supplement beverage was designed to support both muscle and cognitive performance whereas the non-isocaloric standard of care placebo was designed to not promote physical or cognitive health. Both the supplement and placebo were ready-to-drink 8 fluid ounce liquid beverages, peach flavored and were packaged in identical plastic bottles to maintain blinding . The nutritional composition of each beverage is listed in Table Participants were instructed to drink two 8-ounce servings a day for all 12 weeks of the intervention, for a total of 168 beverages throughout the study. Participants were advised to maintain their routine diet throughout the study and to drink one serving of the beverage 30 minutes before the fitness intervention and one serving 1 hour after the fitness intervention on Monday through Friday. On Saturday and Sunday, participants could consume the beverages at any time. The compliance rate for consuming the beverage was high. 47% of the participants consumed all 168 beverages; 51% of the participants consumed between 151 and 168 of the beverages; and 2% of the participants consumed between 141 and 151 beverages.A fitness battery was administered at pre- and post-intervention to assess the efficacy of the exercise training and nutritional supplement. Measurements were taken for 6 domains of physical health and fitness: (1) power (2) strength and endurance 3) mobility and stability (4) blood pressure (5) heart rate (6) lean muscle mass. Table  mobilityA cognitive battery was administered at pre- and post-intervention to assess the efficacy of the exercise training and nutritional supplement. Measurements were taken for 6 domains of cognitive function: (1) short term memory 2) episodic memory (3) fluid intelligence 4) working memory (5) executive function and (6) processing efficiency and reaction time. Table  working  episodic25, folate26, triglycerides27, high-density lipoprotein (HDL), ferritin28, cortisol29 and several fatty acids 30. Lutein was measured in the eye with Macular Pigment Optical Density (MPOD).Blood plasma samples were collected at pre- and post-intervention and analyzed for vitamin B1232). The R packages psych, effsize, compute.es, lsr and BayesFactor were used for the analyses in this manuscript37.All data analyses were conducted using the R Studio interface (Version 1.0.143), which runs on the base R installation  was used to identify the underlying structure of the fitness and cognitive batteries. For fitness, 21 variables from Table Table Table The unimodal exercise plus placebo intervention was compared to the multimodal exercise plus supplement using an ANCOVA model to determine if the addition of the nutritional supplement conferred incremental physical and cognitive benefits. Measures were only tested in the ANCOVA if there was a change from pre- to post-intervention in Table 41. Our study extends this body of work by demonstrating that the combination of a nutritional supplement and an exercise training intervention provided physical health and cognitive gains beyond those from exercise alone. The large sample size of the present study and the large number of assessments across physical fitness, cognition and biomarkers permit five primary conclusions.We conducted an RCT in healthy, fit and active duty Air Force servicemen and women (n = 148) to investigate the efficacy of an exercise training intervention and a novel nutritional supplement for improving physical fitness and cognition. Exercise training interventions are known to improve overall physical health and cognition and we also observed these improvements in the current studyd effect sizes in some cases, and the decisive Bf results, there is clear and compelling evidence that the exercise training intervention was effective and that it improved physical fitness.First, both interventions improved measures of physical fitness. The exercise training plus placebo intervention improved 21 individual measures of physical fitness and the exercise training plus nutritional supplement intervention improved 23 individual measures of physical fitness. Given the huge Cohen’s 41, the exercise training plus placebo intervention drove improvements in cognitive function.Second, both interventions improved measures of cognition. The exercise training plus placebo intervention improved 8 individual measures of cognition. The exercise training plus nutritional supplement intervention effectively improved 11 individual measures of cognition. These results suggest that the combination of a nutritional supplement with the exercise training intervention selectively extended the benefits of the exercise training plus placebo intervention for some of the cognitive outcomes. Consistent with previous research43.The third primary conclusion from the study is that the multimodal exercise training plus nutritional supplement intervention conferred advantages on physical fitness and cognition beyond the effects of the unimodal exercise training plus placebo intervention. Heart rate, lean muscle mass, fluid intelligence reaction time, working memory and processing efficiency all improved more for the multimodal exercise training plus nutritional supplement. Broadly, this is consistent with recent studies demonstrating advantages for multimodal interventions, relative to unimodal interventions, for improving physical fitness or cognition44. In the current study, the exercise intervention decreased inflammation, as measured by ferritin. Vitamin B12, folate, the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) and lutein were consumed daily as part of the nutritional supplement intervention. Increased serum concentrations of these four nutrients were observed in the exercise training and nutritional supplement intervention only. Concentrations of vitamin B12 and folate decreased in the exercise training plus placebo intervention. Concentrations of omega-6 polyunsaturated fatty-acids, which were not part of the nutritional supplement, decreased only for the exercise plus nutritional supplement intervention. Importantly, concentrations of trans, saturated and monounsaturated fatty acids did not change from pre- to post-intervention for either intervention group. Because neither the nutritional formula nor the placebo contained any of these biomarkers, we observe selectivity of biological uptake for the biomarkers that were included in the nutritional formula.Fourth, changes in biomarkers provide further evidence to support the efficacy of the interventions. One inflammatory marker, ferritin, decreased for both intervention groups; but the nutritional supplement did not reduce ferritin levels more than the exercise training intervention. Increasingly, ferritin is recognized as an important marker of inflammation and cell damage; higher levels are associated with worse cognitive functioning45. Younger adult populations show stronger associations between B vitamin supplementation and cognitive improvement46. Therefore, it is reasonable to conclude that B vitamins from the nutritional supplement in the current study are partially responsible for some of the observed improvements in cognitive function in the exercise training plus nutritional supplement group. The nutritional supplement intervention group had increased concentrations of both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Long chain polyunsaturated fatty acids, or PUFAs, like DHA and EPA, are found in high concentrations in the brain’s gray matter. PUFAs in the brain are known to promote neuronal growth and synapse formation and facilitate neural transmission47. Long chain omega-6 polyunsaturated fatty acids are important for brain and cognitive health; but this effect is most pronounced when they are in a balanced ratio with the omega-3 polyunsaturated fats49. Long chain omega-3 and omega-6 fatty acids compete for chemical conversion to various structures and molecules inside and outside cells. In the current study, for the nutritional intervention group, omega-6 concentrations for arachadonic, adrenic, and docosapentaenoic acids all decreased but the omega-3 concentrations of DHA and EPA increased. The increased availability of omega-3’s from the nutritional supplement resulted in a reduction in the ratio of omega-6/omega-3 from 10:1 at the start of the study to 8:1 by the end of the intervention. The ratio remained unchanged at 10:1 from pre- to post-intervention for the exercise plus placebo group. The observed reduction in concentration of several omega-6 fatty acids in the nutritional supplement group was most likely due to the increased availability of omega-3 fatty acids from the nutritional supplement, thereby driving the smaller omega-6/omega-3 ratio which, in turn, contributed to the improved cognitive outcomes for the group receiving the nutritional supplement.These changes in serum concentrations of biomarkers in participants in the nutritional supplement intervention suggest possible mechanisms that might have driven improvements in cognition and physical fitness. B vitamins are critical for brain health50. Furthermore, a dose-dependent relationship exists between the amount of dietary protein and uptake into the body for overall whole-body protein levels and incorporation into muscle fibers51. β-hydroxy β-methylbutyrate (HMB), which was included in the nutritional supplement, synergistically works with protein for increasing lean muscle mass and improves recovery in resistance exercise53. Vitamin D facilitates absorption of calcium in bones and supports muscle growth54. Enhancements to overall physical fitness and health emerged when protein, HMB, protein and vitamin D were taken together as part of an oral nutritional supplement55. And the effects of protein supplementation can be augmented by exercise. Interventions that combine exercise training with nutritional supplements that promote muscle recovery and growth provide the body with necessary building blocks for greater fitness training gains58.Protein, HMB (β-hydroxy β-methylbutyrate) and vitamin D, all of which were included in the nutritional supplement, increase muscle protein synthesis and muscle growth and improve physical fitness. Protein supplementation increases muscle mass and muscle strength59. And different individuals start interventions with different levels of physical fitness and cognitive ability. Accounting for these differential starting points, and possibly using baseline information as a predictor of the possible training gains, could allow further tailoring of intervention protocols and sensitivity of testing to achieve optimal performance gains for individuals. Second, it is not known if interventions like exercise training and a nutritional supplement beverage have upper limits to the physical fitness or cognitive gains achievable, whether at the group or individual level. Dose–response curves would help tease this out. Third, the current study used principal component analysis (PCA) to aggregate physical fitness, cognitive and biomarker measures according to common structure or function. Part of the motivation for using PCA was to amplify the underlying signal and reduce the number of statistical tests required; but there are other statistical approaches which could derive further insights about the efficacy of the interventions. Fourth, it was not possible to include a true control condition because active duty military populations necessarily undergo a certain amount of physical movement in their day-to-day activity. However, Supplementary Note Although the current study provides evidence that the nutritional supplement improved physical fitness and cognition when coupled with exercise training, more research is needed to refine these findings. First, the present study only considered the efficacy of the intervention at the group level. Having demonstrated group level changes to the interventions, follow-up studies should seek to understand the role of individual variability. There is a growing body of research demonstrating that different individuals may need a different number of intervention sessions to achieve the same gain61.The nutritional supplement was a user-friendly way to ensure each participant received equivalent amounts of scientifically proven daily allowances of both macro- and micro-nutrients that supported health benefits. A study using a whole food intervention might confer some advantages over a nutritional supplement, such as greater bioavailability and adsorption of key nutrients; but running a 3-month RCT with whole food intervention presents several challenges. First, the whole food intervention would need to select a variety of foods but still ensure equivalent dosage of key nutrients. Second, in the current study, the non-isocaloric placebo represented a normal diet since those participants had no dietary restrictions. In a whole food intervention, it would be harder to have a true placebo. Third, administering whole food meals is costly (both in terms of time and money) and challenging to track consumption. In the current study, it was easy to watch participants drink two 8-ounce servings of the liquid nutritional supplement in the lab every day; but tracking the consumption of whole food meals is much more challenging. Despite these challenges, future work should attempt to implement a nutritional intervention that includes an isocaloric control. Moreover, researchers should seek to develop nutritional supplements that more closely approximate whole food. To this end, some researchers have begun to elucidate how patterns of nutrient biomarkers, which presumably relate to eating a healthy, whole food diet, enhance cognitive and brain functionIn conclusion, the current study supports the efficacy of a multimodal fitness and nutritional intervention to improve both physical fitness and cognition in Air Force Airmen. Moreover, these changes in the body and brain align with corresponding changes in blood-based biomarkers of nutrition. Our study also demonstrates a wider range of improvements and larger effect sizes due to multimodal training compared to unimodal fitness training alone. While the findings are based on a large sample of Air Force Airmen and demonstrate that physical fitness and cognitive enhancement is achievable, the multimodal lifestyle intervention documented in this study could easily be implemented in other real-world contexts to optimize human performance.Supplementary Information.


KCNJ5 and CACNA1D) and adenosine triphosphatases (ATP1A1 and ATP2B3) highlight the central role of calcium signalling in autonomous aldosterone production by the adrenal gland. The per-SNP BP effect is small for SNPs according to genome-wide association studies (GWAS), and all of the GWAS-identified BP SNPs explain ∼ 27% of the 30%-50% estimated heritability of BP. Uromodulin is a novel pathway identified by GWAS, and it has now progressed to a genotype-directed clinical trial. The majority of the GWAS-identified BP SNPs show pleiotropic associations, and unravelling those signals and underpinning biological pathways offers potential opportunities for drug repurposing. The GWAS signals are predominantly from Europe-centric studies with other ancestries underrepresented, however, limiting the generalisability of the findings. In this review, we leverage the burgeoning list of polygenic and monogenic variants associated with BP regulation along with phenome-wide studies in the context of the mosaic theory of hypertension, and we explore potential translational aspects that underlie different hypertension subtypes.The genetic architecture of blood pressure (BP) now includes more than 30 genes, with rare mutations resulting in inherited forms of hypertension or hypotension, and 1477 common single-nucleotide polymorphisms (SNPs). These signify the heterogeneity of the BP phenotype and support the mosaic theory of hypertension. The majority of monogenic syndromes involve the renin-angiotensin-aldosterone system and the adrenal glucocorticoid pathway, and a smaller fraction are due to rare neuroendocrine tumours of the adrenal glands and the sympathetic and parasympathetic paraganglia. Somatic mutations in genes coding for ion channels ( Page’s “mosaic theory” of hypertension posited that essential hypertension (HTN) is not a single disease, but several different diseases with different origins and development—all causing HTN and its consequences.,KCNJ5) account for 40% of patients with an aldosterone-producing adenoma, whereas 7% are due to mutations in the α-subunit of Na+-K+-adenosine triphosphatase , plasma membrane Ca2+-ATPase (ATP2B3), and L-type Ca2+ channel CaV1.3 (CACNA1D).+ excretion  Family and twin studies have established that BP heritability ranges from 15% to 40% for clinic systolic blood pressure (SBP), and from 15% to 30% for clinic diastolic blood pressure (DBP); for ambulatory BP (sleep), heritability was 69% and 51% for SBP and DBP, respectively.UMOD and PHACTR1/EDN1 genes) that have identified novel pathways with early translational potential because they involve gene products that are the targets for licensed drugs. Then we look at pharmacogenetic interactions that validate pathways targeted by current antihypertensive drugs and explore opportunities for repurposing drugs or tailoring treatment.Despite the plethora of common variants arising from GWAS, the biggest challenge has been linking these variants to a causal mechanism in the BP regulatory pathway. The main reason for this is because GWAS SNPs are selected for screening the genome based on linkage-disequilibrium patterns, and this results in the majority of the signals being in noncoding or intergenic regions. Nevertheless, there are examples of the value of GWAS in identification of novel pathways of BP regulation point to potential subgroups with common underlying pathways that may offer avenues for targeted screening or therapy. We start with 2 SNPs (near UMOD) which is associated with BP and uromodulin excretion. UMOD is almost exclusively expressed in the thick ascending limb of the loop of Henle in the kidney, where 25% of the filtered Na+ is reabsorbed, pointing to a novel sodium-based BP pathway. A potential interaction between uromodulin and the main sodium transporter, NKCC2, in the thick ascending limb of the loop of Henle was established with transgenic mice experiments.,Umod-knockout mice, NKCC2 shows reduced cotransporter activity with consequent greater sodium excretion and a 20 mm Hg lower BP compared with wild-type mice.Umod and hypertensive individuals homozygous for the UMOD increasing allele that the NKCC2 antagonist furosemide had a greater natriuretic and hypotensive effect. This result is currently being tested in a clinical trial  to reposition loop diuretics in the HTN care pathway.A GWAS of BP extremesPHACTR1) gene associated with increased risk of coronary artery disease (CAD) and coronary calcification and decreased risk of migraine headache, cervical artery dissection, fibromuscular dysplasia, and HTN.,EDN1), a gene located 600 kb upstream of PHACTR1.,EDN1 causes vasoconstriction and cell proliferation through activation of ETA receptors (EDNRA) on vascular smooth muscle cells and vasodilatation via release of nitric oxide and prostacyclin (PGI2) through activation of ETB receptors (EDNRB). Endothelin receptor antagonists show BP-lowering properties with both ETA-selective and nonselective drugs .,ClinicalTrials.gov Identifier: NCT02603809) is currently underway to test the efficacy of aprocitentan in resistant hypertension.,ClinicalTrials.gov Identifier: NCT04097314).An intronic SNP in the phosphatase and actin regulatory protein 1  influences 2 or more different phenotypes or traits. Phenome-wide association studies (PheWAS) can identify statistical associations between a single variant and multiple phenotypes and thus reveal pleiotropic associations. PheWAS are usually carried out with the use of a wide range of phenotype data from electronic health records, epidemiologic studies, and clinical trials. Whereas GWAS typically investigates a single phenotype at a time, PheWAS identify all of the traits associated with a genetic variant. With near ubiquitous availability of genome-wide genotyping in most large epidemiologic cohorts and the emergence of biobanks, there is potential to identify pleiotropic SNPs to improve our understanding of the biological functions of a GWAS SNP or identify concealed pathophysiologic connections between traits previously considered as distinct. A look-up of all the 1477 BP SNPs in the GWAS catalogue and PhenoScannerrd cloud , where t,The goal of genomics is to enable precision medicine through a greater understanding of molecular pathways that regulate BP, which can inform new drug development, personalisation of treatment, and ultimately lead to a new taxonomy of HTN.EDNRA and GUCY1A2, respectively) and are candidates for clinical trials for potential extension of their current indication to essential HTN. Valproic acid shows multiple interactions with a range of GWAS loci , indicating that it may be a candidate for repositioning. Nesiritide interacts with NPR3 and is currently not licensed because it failed in a heart failure clinical trial. PheWAS results indicate that pharmacogenes for valproic acid are also associated with adiposity traits and heart rate, pharmacogenes for ET antagonists are associated with CAD, and nesiritide pharmacogene is associated with adiposity, basal metabolic rate, height, lung function, and visceral fat traits. These raise the possibility that new drug development or drug repurposing needs to take into account multimorbid associations for targeting treatment to the right subset of patients. Finally, instead of repurposing drugs, pleiotropic associations open the possibility of multipurposing drugs, for example, prioritising antidepressant drugs that additionally lower BP for patients with hypertension and depression or antiepileptics that also have BP-lowering potential for patients who have both epilepsy and hypertension , which is a mathematical aggregate of risk conferred by all of the SNPs significantly associated with BP. It is important to highlight that the risk information provided by the PRS is different from the risk information from genetic markers of monogenic disorders. The latter is a dichotomous result (either high or low probability of disease), whereas the former provides a wider range of probabilistic risk. In addition, the rare variant genotype points to specific biological impact of the variant, whereas the PRS is an amalgamation of numerous small-effect variants across the genome with no specific pathway implicated. A PRS constructed to use of all of the significant GWAS BP SNPs showed a significant association with stroke, CAD, heart failure, and left ventricular mass, but not for kidney function.,Genomic studies have identified the largest set of SNPs for BP compared with other complex traits. A proportion of these may have translational potential, and the challenge is to identify them. Pleiotropic associations may point to novel underlying pathways or potentially subtypes of essential HTN. Early translational application may be through drug repositioning, followed by new drug development. PRSs look attractive, but their clinical utility needs controlled studies, and the potential ethical impacts of their widespread use exacerbating health disparities need further assessment.10.13039/501100000265Medical Research Council , the 10.13039/501100000274British Heart Foundation , and the BHF Centre of Excellence (RE/18/6/34217).S.P. is funded by the The authors have no conflicts of interest to disclose.


Including individuals with dementia as participants in research studies can be a challenge given the cognitive symptoms experienced, including difficulties with attention, memory, language, and executive functioning and behavioral symptoms, such as agitation and frustration. Researchers, however, have begun to include individuals with dementia as more active participants in the research process. Specifically, individuals with dementia have provided self-report information about their illness experience, including how they cope and manage with their illness and how illness-related strains are related to psychosocial outcomes. Researchers also have starting using self-report data collection protocols instead of proxy-report measures to assess the impact of non-pharmacological interventions developed for individuals with dementia on well-being outcomes such as depressive symptoms, unmet needs, and relationship strain. To date, however, there are no clear guidelines or ‘best practices’ to assist researchers in developing study protocols that facilitate the inclusion and participation of individuals with dementia. This poster will present findings from a systematic literature review of studies that have successfully included individuals with dementia as research participants. Key areas examined include: 1) the focus and type of research question studied; 2) the use and type of cognitive screening tools for determining study eligibility; 3) implementation of qualitative versus quantitative research designs; 4) objective versus subjective measures; 5) data collection tools; 6) psychometric evidence of data obtained; and 7) how data have been analyzed. Findings highlight the importance of collecting data directly from individuals with dementia to further understand the illness experience and assess the impact of non-pharmacological interventions.


Down syndrome (DS) is the most frequent chromosomal abnormality that causes intellectual disability, resulting from the presence of an extra complete or segment of chromosome 21 (HSA21). In addition, trisomy of HSA21 contributes to altered energy metabolism that appears to be a strong determinant in the development of pathological phenotypes associated with DS. Alterations include, among others, mitochondrial defects, increased oxidative stress levels, impaired glucose, and lipid metabolism, finally resulting in reduced energy production and cellular dysfunctions. These molecular defects seem to account for a high incidence of metabolic disorders, i.e., diabetes and/or obesity, as well as a higher risk of developing Alzheimer’s disease (AD) in DS. A dysregulation of the insulin signaling with reduced downstream pathways represents a common pathophysiological aspect in the development of both peripheral and central alterations leading to diabetes/obesity and AD. This is further strengthened by evidence showing that the molecular mechanisms responsible for such alterations appear to be similar between peripheral organs and brain. Considering that DS subjects are at high risk to develop either peripheral or brain metabolic defects, this review will discuss current knowledge about the link between trisomy of HSA21 and defects of insulin and insulin-related pathways in DS. Drawing the molecular signature underlying these processes in DS is a key challenge to identify novel drug targets and set up new prevention strategies aimed to reduce the impact of metabolic disorders and cognitive decline. Down syndrome (DS) is the most frequent chromosomal abnormality that causes intellectual disability, resulting from the presence of an extra complete or segment of chromosome 21 (HSA21). Trisomy of HSA21 is associated not only with intellectual disability but also with several morphological and physiological features. Several regions of the HSA21 contribute to DS clinical manifestations, as suggested by a number of studies performed in human bearing partial duplication . HoweverThe most characteristic trait of DS individuals is disturbance of brain development associated with intellectual disability in the presence of craniofacial abnormalities . FurtherAPP), beta secretase 2 (BACE2), SOD1, and S100 calcium-binding protein B (S100B), among others -peptide is considered one of the major toxic players in the early onset of AD neuropathology in DS population , such as the deposition of senile plaques and neurofibrillary tangles . After tg others . The ovepulation . It is wpulation . Moreovepulation . The exapulation .Vingt Ans Après,” he explained how the one carbon cycle could be involved in the pathogenesis of intellectual disability in subjects who do not have a gross anatomic defect of the brain, and he asserted: “the goal is to figure out where a link between mental deficiency and trisomy 21 should be sought.”Dr. Jerome Lejeune was the first to hypothesize that DS could be considered a “metabolic disease.” This is highlighted by To confirm the “Lejeune hypothesis,” a number of alterations on metabolite concentrations have been observed in the blood of individuals with DS compared with age-matched control subjects, in particular alteration of a number of amino acids . Specifi2S is responsible for mitochondrial deficits, in particular by inhibiting complex IV , which interferes with the pyridoxal phosphate in the catalytic site, restored Complex IV activity and ameliorated mitochondrial electron transport and cell proliferation. Similar effects were also obtained by CBS normalization by siRNA (2S)] and circulating sulfhemoglobin (addition of H2S to hemoglobin) levels have already been detected in subjects with DS (The levels of these metabolites seem to be strongly associated with triplication of CBS . As recemplex IV . These rby siRNA . Moreove with DS .FTCD) is another gene located on the long arm of HSA21, which encodes an enzyme that participates in histidine and folate metabolism, both essential for purine, pyrimidines, and amino acids biosynthesis. In addition, aberrant metabolism of adenosine, homocysteine, and folate was also observed in DS , a regulatory enzyme in glycolysis as it catalyzes the phosphorylation of fructose-6-phospate to fructose-1, 6-bisphosphate . InteresFurthermore, DS subjects are characterized by an impaired lipid metabolism, although this aspect has been less investigated. Published studies report that DS children show higher levels of circulating cholesterol, low-density lipoproteins (LDL), and triglycerides with respect to age-matched controls . Less faIn the brain, a lipidomic study performed by As a whole, the picture that emerges from these studies unravels a pathological metabolic phenotype of DS contributed by a number of different genes. Within this scenario, defects of mitochondrial function contribute to a general loss of cellular functions, most of which strictly depend on ATP availability .2.In catabolic tissues, mitochondria are the key organelles responsible for energy production that sustains a plethora of intracellular functions, which as a whole approximately consume about 90% of oxygen to generate ATP through oxidative phosphorylation (OXPHOS) . MitochoA growing number of studies also demonstrated that loss of mitochondrial structure and function, which is associated with increased ROS production, contributes to DS pathological phenotypes . This isReduced rate of energy metabolism due to mitochondrial dysfunction significantly impairs neuronal functions, as well as neuronal development and survival . Indeed,Vacca et al. analyzed mitochondrial function in fibroblasts and lymphoblastoid cells from DS subjects and found deficits in the OXPHOS system at multiple levels such as the complex I activity, the ATP synthase, the ADP/ATP translocator, and the adenylate kinase enzyme, ultimately leading to significant energy deficit and increased ROS production in mitochondria , 2011. ASeveral bioactive compounds display protective effects on mitochondrial signaling pathways, mitochondrial biogenesis, and respiration . Both naAmong the metabolic alterations observed in DS subjects, dysfunctions of insulin signaling and related pathways are of interest. Although insulin or proteins belonging to the insulin signaling pathway are not encoded by genes located on HSA21, insulin signaling is at the crossroad among a number of intracellular events driving cell metabolism in terms of glucose, fatty acids, and proteins synthesis/utilization .The frequency of metabolic diseases associated with defects of insulin and insulin-related pathways develop with high frequency in DS subjects. Furthermore, over the past few years, it has become clear that insulin also has profound effects in the central nervous system, where it regulates key processes such as energy homeostasis and neuronal functions .In the next sections, we summarize the accumulating evidence on peripheral and brain defects of insulin signaling and related pathway in DS. Current findings along with pathogenic factors and consequence in terms of disturbances are discussed.Obesity and subsequent metabolic disorders show higher prevalence in adult population with DS compared to individuals without DS. Among the weight-related and metabolic disorders present in DS individuals, glycemic dysregulation presents involving impaired fasting glucose, diabetes mellitus, dyslipidemia, and metabolic syndrome. DS individuals have higher body mass index (BMI) and body fat percentage (%BF) compared to age- and sex-matched persons without DS . InteresAmong the hormonal regulators of fat accumulation and energy balance, leptin levels in plasma correlate with adiposity in the general population but also in DS children and adolescents . DS fetuHowever, an inherent difficulty of human studies when trying to extract conclusions of metabolic features in different cohorts is the existence of confounding factors such a lifestyle and regional nutrition habits and preferences. Animal models provide a suitable alternative to investigate the impact of the HSA21 genes on the metabolic alterations in DS controlling for environmental factors. Recently, we investigated the effect of the trisomy in the metabolic-inflammatory axis and their relation with energy expenditure, energy intake, and fat accumulation in Ts65Dn mice, a partial trisomy DS model . Ts65Dn There is also a higher prevalence of diabetes in DS individuals compared to the general population . Fetal hRCAN1, since transgenic mice overexpressing the human RCAN1-1 isoform present age-dependent hyperglycemia, impaired glucose tolerance, hypoinsulinemia, reduced β-cell secretion, reduced β-cell number, decreased insulin granule filling, and reduced mitochondria size in β-cell along with an aberrant mitochondrial reactive oxygen species production  (356)-GSK3β in the white adipose tissue, and downregulation of adipogenic proteins , Kunej and collaborators describe in the general population a genetic association between quantitative traits loci (QTL) for body weight (BW276_H) and these seven loci located in the HSA21 .Certainly, besides the overexpression of coding and non-coding regions of the HSA21, genes located in other chromosomes whose expression is dysregulated could also take part in the DS phenotypes. This genome-wide transcriptional deregulation could be due to epigenetic modifications. In trisomy 21 discordant twins as well as the Ts65Dn/WT mouse cells, the chromatin modifications produced by nuclear compartments of T21 cells affect the overall . AlternaINSULIN and CHRNA1genes  relevant for this embryonic process. Consequently, Dyrk1A-overexpressing mice show significantly thicker but less functional thyroids, as shown by the lower T4 hormone excretion and the tendency to have increased plasma TSH  and associated effects, the insulin resistance phenomenon by itself plays a crucial role during the preclinical stage of AD . This isMechanisms identified to prompt the development of brain insulin resistance  include,From a molecular point of view, the activation of insulin signaling requires the binding of insulin to IR, which auto-phosphorylates and promotes the phosphorylation of the insulin receptor substrate 1 (IRS1) on specific Tyr residues . Once activated, IRS1 works as scaffold protein driving the activation of the two main arms of the insulin signaling pathway: (1) the phosphatidylinositol 3-kinase/3-phosphoinositide-dependent protein kinase 1/protein kinase B (PI3K/PDK1/Akt) and (2) the mitogen-activated protein kinase (MAPK) pathways . InsteadStudies performed in human and mouse models of AD highlighted the role of inflammation and in particular the inflammatory cytokine tumor necrosis factor alpha (TNF-α), which leads to the activation of specific kinases   and endoin vitro , in DS with respect to age-matched control . Of noteAs reported by our group, intranasal rapamycin , thus contributing to worsening a pre-existing condition defined on a genetic background. This aspect is fascinating especially in light of the role of insulin signaling pathway in regulating energy metabolism.DS subjects represent a unique population that needs to be carefully followed during the entire course of their life. One of the main defects caused by trisomy of HSA21 is metabolic defect. Indeed, DS individuals show a pattern of metabolic defects that contribute to increase the risk of developing chronic diseases such as diabetes or AD-like dementia. This is a crucial aspect considering that metabolic diseases characterized by insulin resistance, such as obesity and diabetes, are associated with a progressive cognitive decline as well as with a risk of developing dementia in the general population. Keeping this in mind, cognitive decline occurs in DS because their genetic background could be further aggravated by the onset of metabolic disorders, which could in turn foster the development of AD in DS patients. Despite knowledge concerning the molecular mechanisms potentially involved in the progression of peripheral and brain metabolic disorders in DS, early alterations are still unknown and require further investigations. Indeed, identifying these alterations during their initial stage could have the advantage of being caught early enough to stop/delay their progression. In this picture, defects of insulin signaling seem to occur early in life at both peripheral and brain levels and persist with aging in DS. Furthermore, at the cellular level, the dysfunction of insulin signaling pathway crosses with dysfunctions of proteins encoded by genes on HSA21  , 2015. FIn conclusion, drawing the molecular signature underlying alterations of insulin signaling in DS is a key challenge to identifying novel drug targets and set-up new prevention strategies aimed to reduce metabolic disorders as well as their impact on cognitive decline in DS.All authors conceived the work, took part to the scientific discussion, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It is a major cause of irreversible blindness worldwide. Primary glaucoma is classified into three major forms based on the anatomy of the anterior chamber drainage angle of the eye and the age of onset: primary congenital glaucoma (PCG), primary open angle glaucoma (POAG), and primary angle closure glaucoma (PACG). In glaucoma patients with increased IOP, the increase is thought to be mainly due to impaired drainage of aqueous humor from the anterior chamber.Glaucoma is a heterogeneous group of optic neuropathies characterized by a specific pattern of optic nerve degeneration and visual field loss that is usually accompanied by increased intraocular pressure (IOP).,5,6,7 POAG and PACG are generally considered complex multifactorial disorders. Several POAG-causing genes have been identified, but mutations in these account for disease in less than 10% of patients.,11,12,13The etiology of all forms of glaucoma includes a genetic component as evidenced by variable prevalence in different ethnic groups, observations on familial clustering, and results of pedigree and sib-pair studies. For PCG, which is usually a monogenetic Mendelian disease, three causative genes have been identified.,15,16 Most PACG patients are from Asia, particularly China, Mongolia, Singapore, and India.Compared to the other forms of glaucoma, there is much less definitive genetic data pertaining to PACG. This likely reflects the contribution of multiple genetic and perhaps environmental factors that affect various anatomical and functional features associated with PACG. PACG is an important public health entity. It is estimated that 15.7 million individuals in the world are affected with PACG. It is projected that 21 million will be affected by 2020, and that PACG by that time will cause bilateral blindness in 5.3 million people.COL18A1 which encodes collagen type XVIII was identified as a gene that affects angle closure in patients of three unrelated families. The inheritance pattern of angle closure causing mutations in COL18A1 was autosomal dominant. It appears that mutations in this gene may cause angle closure in the fourth decade of life or later. Furthermore, COL18A1 mutations are not expected to be a common cause of angle closure-related phenotypes. The significance of having identified COL18A1 as a potential PACG-causing gene lies in emphasis on the importance of collagens and the extracellular matrix in glaucoma pathology.Ultimately, the defining feature of PACG in individuals with glaucomatous optic nerve damage is iridocorneal angle closure. Recently, ,23,24,25 However, these findings are not generally considered definitive, and putative roles of genes suggested by some studies were not confirmed in independent studies. Factors known to be associated with PACG include hyperoptic refractive error, shallow anterior chamber, thick crystalline lens, short axial length, small corneal diameter, and narrow iridocorneal angle.,27,28 Clearly, some of these also associate with each other. In an article published in this issue of the Journal of Ophthalmic and Vision Research, the authors relied on transcriptome data pertaining to eye anterior segment tissues to select five single nucleotide polymorphisms (SNPs) whose genotypes may be associated with PACG among patients of Northeast Iran.,30,31 Interestingly, one of the SNPs for which an association with PACG was reported is positioned in a gene (FERMT2) that encodes a component of the ECM. Results of an association study that included tens of thousands of patients and controls had also implicated the same gene with respect to PACG.In pursuit of identifying genes that contribute to PACG disease, association studies and candidate gene studies have been performed and the results of these have implicated possible roles for several genes.,34,35,36 Eight loci were identified in one or both of two recent large genome-wide association (GWA) studies on PACG patients, and COL11A1 that encodes one of the alpha chains of type XI collagen was one of the genes identified in both of the studies.,20 An SNP in COL1A1 was associated with increased risk of myopia in Japanese and Chinese individuals.,38 A GWA study of PACG in a dog breed identified COL1A2 as a susceptibility locus. And of course, COL18A1 was identified as a gene that affects iridocorneal angle closure in humans. In addition to collagens, genes with roles in the extracellular matrix and also associated with glaucoma include MMP-9, MTHFR, LTBP2, CYP1B1, and SPARC. It has been suggested that inter-individual differences in tolerance to IOP as reflected in glaucoma diagnosis with normal tension in some and ocular hypertension without glaucoma in others reflect variations in biomechanical properties of the extracellular matrix of relevant ocular tissues.There exists accumulating evidence that implicates various collagens specifically and the extracellular matrix more generally in the pathogenesis of glaucoma.


Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions. G protein-coupled receptor kinases (GRKs) encompass seven protein isoforms that belong to three different subfamilies: the visual subfamily including GRK1 and GRK7, the GRK2 family including GRK2-3 and the GRK4 family composed of the GRK4, -5 and -6 isoforms. GRK2-3 and GRK5-6 proteins are ubiquitously expressed in cells and tissues, while GRK1 and GRK7 are localized in the retina, and GRK4 is mainly localized in the testis [CXCR4 gene, these observations support the critical role of GRKs in controlling this chemokine receptor’s functions.Several genetic mouse models have provided clues to the importance of GRKs in physiology, as extensively discussed elsewhere . ConcernFrom the molecular perspective, GRKs are thought to primarily control immune processes by regulating cell migration in both homeostatic and inflammatory conditions through their action on chemokine receptors that are master regulators of immune cell trafficking. In humans, 18 of 23 chemokine receptors are GPCRs that trigger canonical pathways upon agonist activation, leading to GRK-mediated phosphorylation of the receptor intracellular domain and subsequent binding of arrestins, a physiological feedback mechanism of homologous desensitization that rapidly uncouples the receptor from G proteins ,33. WhilThe chemokine system encompasses chemokine ligands, and their 7TM receptors  generally display redundancy and binding promiscuity, with the notable exception of CXCL12 and its typical and atypical receptors, CXCR4 and ACKR3, respectively . These rA large body of literature has established the critical importance of GRKs in initiating the process of desensitization of chemokine receptors in a kinase-dependent manner via arrestin recruitment, as summarized in Taken together, these results prompted several hypotheses with regard to the regulatory role of GRKs in chemotaxis. Different GRKs might be recruited to the receptor upon chemokine binding and interact with distinct domains of the receptor, according to the availability of the other components of the signalling pathways that are activated ) a. This cThe phosphorylation barcoding hypothesis proposes that each GRK preferentially phosphorylates a limited number of distinct serine and threonine (Ser and Thr) residues on a particular GPCR, thus creating a barcode. While other protein kinases  mediate agonist-dependent phosphorylation of activated GPCRs, their roles are beyond the scope of this review ,47,48. TSeveral reports support the possibility of these mechanisms for CXCR4, the broadly expressed G protein (mostly the inhibitory Gi family member)-coupled receptor of the CXCL12 chemokine. This pair, which exhibits a high degree of conservation throughout evolution from jawless fish to humans, is essential for embryogenesis, including proper haematopoiesis, and plays nonredundant functions in immune homeostasis ,50. An oThe disruption of the phosphorylation barcode through mutations in the C-ter of a chemokine receptor may have serious pathological consequences, as illustrated by WHIM syndrome. This syndrome is the first example of a human disorder mediated by dysfunctions of a chemokine receptor, namely CXCR4, and is mostly linked to inherited heterozygous autosomal dominant mutations in the CXCR4 gene . All eleThis organization would provide an explanation of why and how the different WHIM-associated mutations translate into broad dysfunction downstream of the CXCL12/CXCR4 axis. Along with this phosphorylation pattern, investigations of the pathogenesis of WHIM syndrome permitted us to assign a major role to GRK3 in regulating CXCL12-induced desensitization of CXCR4. Indeed, on the one hand, the impairment of CXCL12-induced CXCR4 internalization harboured by cells derived from patients with WHIM lacking CXCR4 mutations  was attrThe enhanced and prolonged CXCL12 responses featured in WHIM syndrome  may also result from aberrant activation of arrestin-dependent pathways, as originally revealed by the impaired migration of leukocytes from β-arrestin 2 knockout mice in response to CXCL12  and furtFinally, in light of the multiple protein partners reported to physically interact with GRKs or to display concurrent regulation of their expression with GRKs , The second receptor for CXCL12 is a member of the smallest subgroup of atypical chemokine receptors, 4, in humans; these receptors do not signal through G proteins and, rather, regulate GPCR-driven chemotaxis by tuning chemokine concentrations in tissues  . AccordiImportantly, the scavenging function of ACKR3 has also been presented as a mechanism that is notably involved in the proper positioning of neurons in the developing mouse brain ,101,102 Among the chemokine receptors, CCR7 provides insights into how different chemokine ligands, namely CCL19 and CCL21, contribute to receptor functions by activating distinct signalling pathways through the generation of selective conformational changes of the receptor associated with a distinct GKR-induced C-ter phosphorylation barcode and, thus, the differential recruitment of effectors a. Both CThe most prominent structural difference between CCL19 and CCL21, which otherwise share the typical tertiary structure of chemokines, is the highly charged C-ter domain of CCL21 that confers this chemokine with the capacity to interact with the cell surface and extracellular matrix-associated glycosaminoglycans (GAGs), while CCL19 is mostly soluble ,121,122.-/- neutrophils [-/- lymphocytes [These findings were extended to CXCL12/CXCR4-induced persistent directional neutrophil chemotaxis, whereby GRK3-deficient neutrophils remained able to spatially and temporally sense stable CXCL12 gradients . In additrophils  but decrphocytes . Regardiphocytes ,129. Forphocytes ,130.The CXCL8 chemokine is the ligand of two highly homologous chemokine receptors, CXCR1 and CXCR2 b. CXCR1 The molecular connections between chemokine receptors and GRKs have prompted the question of how their relative expression is regulated, a question that has been most particularly addressed in relationship with trafficking neutrophils in the context of inflammation. We will outline studies that advanced our understanding of the mechanisms controlling the activities of GRKs and their effects on neutrophil guidance in health and disease, with a focus on CXCR4 functions, in light of WHIM-associated neutropenia and the deregulation of GRK expression in pathological conditions and their interplay with non-GPCRs.As mentioned, the GRK2, 3, 5 and 6 isoforms are broadly expressed in leukocytes, although with differential transcriptional expression patterns among cell lineages and cells with different developmental and activation statuses. Most notably, the Immunological Genome Project  has provCompelling evidence indicates a critical role for CXCR4 desensitization in the processes controlling the residence of neutrophils in the BM and their release into the blood. These homeostatic processes are controlled by the balanced interplay between two chemokine systems , where CXCR4 expressed by BM neutrophils is desensitized and internalized in response to high local concentrations of CXCL12 within BM microenvironments, while low levels of CXCR2 ligands expressed by endothelial cells recruit neutrophils out of the retentive CXCL12 domains for entry into the blood circulation in a CXCR2-dependent manner ,145.Therefore, the neutropenia that characterizes patients with WHIM ,60 is unNeutrophils are short-lived cells in the bloodstream that are cleared from blood and eliminated upon infiltration into the BM, liver and spleen , as wellRecent analyses of long-range neutrophil migration in 3D chamber devices have provided insights into the striking interplay between the strengths of chemoattractant gradients and the desensitization of chemokine receptors in a GRK-dependent manner . This meThe spatial and temporal contributions of chemokines and/or other chemoattractants  to the migratory modes of neutrophils either during homeostasis or in the course of the transmigration of neutrophils through the endothelium into inflamed tissues has been recently discussed by Lämmermann and Kastenmüller . SeveralIn other pathological contexts, GRK2 is a very relevant signalling hub, such as in heart failure, where GRK2 protein expression levels are increased at late stages after myocardial infarction  but are The past two decades witnessed conceptual advances in identifying the multifaceted nature of GRKs. However, in addition to the foundational paradigm that GKRs participate in receptor desensitization, studies of their roles as positive regulators of the chemotactic responses of immune cells revealed some conceptually novel leads, including the importance of the phosphorylation barcoding of a receptor in guiding cellular physiological outcomes and modes of migration. These recent findings have raised important questions regarding the diversity of both chemoattractant signals encountered by immune cells and their GPCRs: How do GRKs regulate chemokine/chemokine receptor systems? How do different GRKs operate in concert or sequentially to mediate GPCR functions? How do networks of chemoattractants, receptors and GRKs respond to metabolic or circadian clocks? Information on the mechanisms regulating the GRK levels and activation during homeostasis and inflammation is lacking. In addition, the reported roles of several chemokine receptors in cell proliferation and tissue regeneration have prompted the question of how GRKs are associated with these critical functions. In line with this, studies investigating how atypical chemokine receptors are regulated by GRKs and how their expression, notably by the endothelium, contributes to environmental cues will provide further insights into how this complex network operates. A second important conclusion from these studies is that GRKs appear to be neither promiscuous nor interchangeable with regard to a particular chemokine/chemokine receptor system: all various functional and physiological outcomes might depend on changes in the expression or the activation of a specific GRK. Future studies are required to define whether and how these changes occur, along with the development and activation status of leukocytes in relationship to the tissue environment. In addition, deciphering the mechanisms that fine-tune the expression levels of GRKs in inflamed sites will increase our understanding of how adversely or positively GRKs affects disease development. All of these questions are also particularly important for the development of future therapeutic strategies.


Candida infection in 1969. The ability to detect genetic variants and mutations that lead to rare or subtle susceptibilities has improved with techniques such as single nucleotide polymorphism (SNP) microarrays, whole exome sequencing (WES), and whole genome sequencing (WGS). Still, these approaches have been limited by logistical considerations and cost, and they have been applied primarily to Mendelian impairments in anti-fungal responses. For example, loss-of-function mutations in CARD9 were discovered by studying an extended family with a history of fungal infection. While discovery of such mutations furthers the understanding of human antifungal immunity, major Mendelian susceptibility loci are unlikely to explain genetic disparities in the rate or severity of fungal infection on the population level. Recent work using unbiased techniques has revealed, for example, polygenic mechanisms contributing to candidiasis. Understanding the genetic underpinnings of susceptibility to fungal infections will be a powerful tool in the age of personalized medicine. Future application of this knowledge may enable targeted health interventions for susceptible individuals, and guide clinical decision making based on a patient's individual susceptibility profile.Fungi are ubiquitous. Yet, despite our frequent exposure to commensal fungi of the normal mammalian microbiota and environmental fungi, serious, systemic fungal infections are rare in the general population. Few, if any, fungi are obligate pathogens that rely on infection of mammalian hosts to complete their lifecycle; however, many fungal species are able to cause disease under select conditions. The distinction between fungal saprophyte, commensal, and pathogen is artificial and heavily determined by the ability of an individual host's immune system to limit infection. Dramatic examples of commensal fungi acting as opportunistic pathogens are seen in hosts that are immune compromised due to congenital or acquired immune deficiency. Genetic variants that lead to immunological susceptibility to fungi have long been sought and recognized. Decreased myeloperoxidase activity in neutrophils was first reported as a mechanism for susceptibility to  We are constantly exposed to fungi capable of causing disease. Every human is colonized with a commensal fungal mycobiome  was first reported as a case series of opportunistic fungal infections in homosexual men  continues to be a threat. PIDs are inherited defects in the immune response; depending on the affected pathway(s), PIDs have different severity, onset, and risks of infection by certain groups of organisms. Perhaps the most dramatic PID is Severe Combined Immunodeficiency (SCID), a condition where the absence of an adaptive immune response uniformly leads to overwhelming infections and death in the first few years of life in the absence of stem cell transplantation. SCID was first described in 1950 and soon recognized as heritable Buckley, .CARD9 have been associated with autosomal recessive inheritance of susceptibility to invasive infections by fungi, including Candida spp., dermatophytes, and fungal plant pathogens . They found that donors were either exclusive carriers of one strain, predominant carriers of one strain with occasional other strains recorded, or “random” carriers who did not appear to have a predilection for one strain  of fungal infection. Abdel-Rahman et al.  studied CLEC7A-Y238X, an early stop codon variant that impacts recognition of fungal β-glucan by the receptor Dectin-1, was reported in a Dutch family where all were affected by onychomycosis , and have not discussed an association with superficial dermatophytosis. Other studies of onychomycosis propose a role for variation at the Human Leukocyte Antigen (HLA) complex, which is involved with presentation of extracellular antigens to T cells and initiation of adaptive responses  due to Dermatophytosis is one of the most common infectious diseases globally, often presenting as self-limiting superficial infections. Environmental factors strongly influence the development of disease. Still, carriage of dermatophytic fungi is correlated with illness, and this is influenced strongly by a small number of genes involved in leukocyte function and other processes , in excess of 3 million cases per year of mucosal candidiasis , and about 750 thousand cases per year of invasive candidiasis and candidemia  is increasing   is a soluble CLR that activates the complement cascade, facilitating opsonophagocytosis of fungi   donors  or CARD9 . Decreased activity of NLRP3, associated with NLRP3 allele 7, is associated with RVVC . In support of their reported association, the authors noted increased IL-1β production in response to C. albicans for NLRP3 genotype 12,9 vs. 12,12  in Caucasian women  and esophageal candidiasis (OEC) are closely associated with the immunocompromised state. Together, these conditions affect about 5% of HIV-positive patients on antiretroviral therapy and 20% of those with low T cell counts  , TLR2, TLR4, TIRAP, CARD9], cytokine production (CASP12), and autophagy (ATG16L1 and IRGM)  and disseminated candidiasis are feared complications of immunosuppression. Mortality rates of over 20% have been reported , TLR adaptors , and FCγRs  , generation of reactive oxygen species (CYBA), or chitin breakdown (CHIT1) , a cytokine receptor (IL12RB1), and a gene involved in cytokine processing (CASP12)  in the context of neutropenia. Both a protective haplotype and a risk haplotype were identified in MDA5 in association with candidemia  CD58, TAGAP, and the LCD4A-C1orf68 locus  are cytokine quantitative trait loci (cQTL) for IL-6   are recognized by dimeric receptors composed of the five receptor subunits in the IL-17R family (IL-17RA through IL-17RE), with an intracellular adaptor (ACT1)  suffer from invasive fungal disease due primarily to filamentous fungi, although invasive candidiasis is also reported   deficiency. MPO deficient cells are unable to generate HOCl, although generation of an oxidative burst is unaffected   worldwide , and at-risk patients include those with COPD, sarcoidosis, and chronic pulmonary infections that cavitate such as tuberculosis , PRRs , and the genes DENND1B, PLAT (plasminogen activator), and VEGFA  is associated with the development of CPA in a cohort of Chinese COPD patients , and almost a quarter of HSCT patients develop IA in some studies , or recipients (S100B)  or CD209 (DC-SIGN) variants are associated with the development of IA in patients with hematologic malignancies ; this is thought to be a function of exposure to an especially large inoculum in many of the cases (Castillo et al., B. dermatitidis; MyD88−/− mice succumb to lethal infection even by an attenuated vaccine strain that is non-lethal in wild-type mice (Wang et al., Studies in a mouse model of blastomycosis have revealed a critical role for early responses by lung epithelial cells (Hernandez-Santos et al., Recently, work has addressed the genetic underpinning of health disparities in rates of blastomycosis in otherwise healthy individuals. A homozygosity mapping approach was used to study 9 Hmong blastomycosis patient genomes, and candidate susceptibility variants were identified on the basis of their rarity in European populations and other features that may indicate that a given variant influences susceptibility (Merkhofer et al., A genetic basis for susceptibility to endemic dimorphic fungi has long been hypothesized, due to their propensity for causing disease in otherwise healthy people, and to their predilection for people of certain genetic backgrounds. Extensive experiments in mouse models have highlighted similarities and differences in the immune responses to this diverse group of pathogens. Only in the past few years have studies in patients begun to reveal those processes that are critical to human immunity. The role of Th1 and Th17 responses is evinced by the severe infections seen in patients with monogenic deficits in those pathways. More recently, studies of disseminated coccidioidomycosis and pulmonary blastomycosis have revealed subtle variants in these pathways. The identification of these variants represents progress toward understanding the genetic underpinnings of observed health disparities in these pathogens.While an increasing number of mutations and variants are reported to confer risk of fungal diseases, only PIDs and Mendelian susceptibility variants are currently clinically actionable. This is partially because there are substantial challenges in translating these findings to clinical applications. By necessity, most studies of susceptibility to fungal diseases have limited their donor population to one ethnic/racial background, which may limit generalizability of findings or, at the very least, necessitate extensive validation of variants in diverse patient populations. Still, the availability of next-gen sequencing technologies may soon make it feasible to incorporate testing for subtle risk variants into clinical practice, and guidelines will be developed for how to incorporate an individual's susceptibility profile into patient care. Future efforts may seek to develop tools to stratify patients by risk based on susceptibility variants; for example, IA susceptibility variants could be incorporated into a tool to stratifying potential HSCT donors by risk of invasive molds.RM and BK conceived the content of the manuscript together. RM drafted much of the manuscript. BK contributed to portions, and reviewed and edited the entire manuscript. RM and BK jointly finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Patients with, or who develop, metastatic breast cancer have a 5-year relative survival of about 25%. Endocrine therapy clearly improves outcomes in patients with estrogen receptor-positive breast cancer. In the metastatic setting, the primary goal of treatment is to maintain long-term disease control with good quality of life. Rarely, exceptional responders achieve durable disease control, and potential cures cannot be ruled out.We report the case of a 39-year-old woman with primary breast cancer and associated synchronous bone metastases, who experienced a disease response of 12 years with hormonal therapy as maintenance after first line chemotherapy, with a good toxicity profile.The patient was diagnosed with estrogen receptor + human epidermal growth factor receptor 2 (HER2)− metastatic breast cancer with synchronous bone metastases.This patient was treated with chemotherapy for 6 cycles as a first-line therapy following by endocrine treatment given as a maintenance therapy.Our patient experienced a progression-free survival >12 years with an exceptionally good quality of life.Our anecdotal experience highlights the existence of exceptional responders among patients with hormone receptor-positive metastatic breast cancer, who achieve clinical remission and durable disease control with endocrine therapy. Being able to identify these patients could help in the selection of the best treatment option among the many available. In patients with estrogen receptor (ER)-positive breast cancer, endocrine therapy clearly improves outcomes and is considered the first-choice treatment for advanced disease, apart from the rare cases of visceral crisis. Adding a cyclin-dependent kinase (CDK) 4/6 inhibitor further improves outcomes. Premenopausal women with HR-positive metastatic breast cancer should be offered ovarian suppression or ablation plus a further endocrine agent, with or without a CDK4/6 inhibitor. We report the case of a 39-year-old woman with primary breast cancer and associated synchronous bone metastases, who experienced exceptional response achieve durable disease control, with hormonal therapy as maintenance after first line chemotherapy, with a good toxicity profile.Breast cancer is the most prevalent cancer in women, in the developed world. Long-term survival outcomes are related to disease stage at presentation. Patients with, or who develop, metastatic breast cancer have a 5-year relative survival of only about 25%,22 intravenously [i.v.] on day 1), cyclophosphamide (500 mg/m2 i.v. on day 1) and weekly docetaxel , with cycles repeated every 21 days, achieving a partial response after 3 cycles. In October 2006, after prior panoramic radiography of the jaw, zoledronic acid 4 mg i.v. every 4 weeks was added to chemotherapy. A CT scan performed after 6 cycles of chemotherapy confirmed a partial response of the disease. A bone MRI showed a major reduction of the neoplastic involvement with substitution of osteoblastic for the osteolytic lesions, especially at D6 vertebral body , infiltrating carcinoma of the breast, with ductal predominant pattern, was made. The major tumor diameter was 35 mm, there was peritumoral vascular invasion, and 1 out of 33 axillary lymph nodes was metastatic. Immunohistochemistry showed that 70% of tumor cells were positive for ER and 10% for progesterone receptor (PgR), the HER2-neu score was 0 and the Ki67 labeling index was 35%. According to the pathologic stage pT2 pN1a, she performed postoperative work-up including chest X-ray and abdominal ultrasound, that resulted negative for distant metastases. A bone scan and a confirmatory magnetic resonance imaging (MRI) were also performed and showed 2 lesions at the sixth and eleventh dorsal vertebral bodies  and the high proliferative index (Ki67 35% in the primary tumor), despite only the bones were involved by the presence of metastases. Given that in the metastatic subset of patients the primary goal is to maintain long disease control with good quality of life, after 6 cycles of chemotherapy and evidence of disease response we switched to a hormonal treatment. According to the literature data, that show better progression-free and overall survival in patients receiving combined therapy with Luteinizing Hormone-Releasing Hormone (LHRH) analog plus tamoxifen, respect to patients treated with tamoxifen alone or LHRH analog alone, we chose this type of treatment for our young patient.,10 Given the bone metastatic involvement, our patient started also treatment with zoledronic acid. Bisphosphonates impact on the rate of skeletal events, reducing the risk of pathological fractures and the need for radiotherapy to control pain and to avoid medullary compression. Before starting this therapy, given that these drugs may be associated with the risk of osteonecrosis of the jaw, it is strongly advised to perform a dental examination for an oral hygiene session and an orthopantomography, even if in recent prospective studies the incidence of this complication was very low (1%–2%). To date, the optimal duration of bisphosphonates therapy has not been definitely established. The National and International Guidelines indicate to administer therapy for a period of at least 1 to 2 years, up to the entire duration of therapy for metastatic breast cancer, until there is a substantial decline in the patient's conditions. However, our patient permanently interrupted zoledronic acid due to the need to perform invasive dental procedures, given the possible onset of osteonecrosis of the jaw, in the context of an improved bone metastatic disease after approximately 12 months of therapy. Our report exemplifies a case of exceptional response with long-term survival of a patient with oligometastatic HR-positive breast cancer. The concept of metastatic cancer as a chronic disease has now been embraced by both physicians and patients. In rare cases, survival extends beyond what usually expected, raising the issue of curability of metastatic disease, as pointed out in recent reviews. Reports from chemotherapy studies conducted for metastatic breast cancer, such as those from the MD Anderson or from the Eastern Cooperative Oncology Group, suggest that a small fraction of patients, perhaps in the order of 1% to 2%, may achieve long-term (≥10 years) disease-free survival.,17 This occurs more frequently in cases of oligometastatic disease, where up to 50% of the patients have been reported to achieve long-term survival after systemic therapy, sometimes combined with a local therapy.,19 We cannot obviously establish if the chemotherapy, administered at our patient as first-line treatment, contributed in some way to the achievement of a long-term survival. The Cochrane's review comparing the effects of chemotherapy alone with endocrine therapy alone in metastatic breast cancer found that while initial treatment with chemotherapy rather than endocrine therapy may be associated with a higher response rate, the 2 initial treatments had a similar effect on overall survival. The impact of chemotherapy in case of oligometastatic disease, a condition closer to that of micrometastatic disease in radically operated breast cancer, cannot in principle be excluded, but there are no data supporting this hypothesis. Radiation therapy is delivered with some frequency to oligometastatic sites in patients with breast cancer. Although there are some hints of potential impact on long-term disease control, and although advanced techniques, such as conformal radiotherapy and hypofractionation, may reduce toxicity and improve the feasibility of treatment, demonstration of benefit from prospective randomized clinical trials is still lacking. Given the lack of symptoms and of risks of skeletal events, we did not prescribe radiotherapy to the metastatic lesions. A further question arising is if it is possible to hypothesize a treatment interruption after a long period of response to endocrine treatment for advanced breast cancer. Literature reports of treatment interruption after durable complete response can be found for trastuzumab,–24 with some patients maintaining prolonged progression-free survival also after trastuzumab interruption, although data are quite scanty and this strategy is not endorsed by current guidelines. To our knowledge, there are no reports of successful experiences involving suspension of endocrine therapy after prolonged remission in metastatic breast cancer and continuing endocrine therapy until disease progression is the current standard of care. Intermittent treatment proved to be inferior to continuous treatment in metastatic breast cancer, and the Dutch Breast Cancer Research Group “Stop & Go study” study concluded that intermittent first-line treatment cannot be considered in patients with HER2-negative advanced breast cancer. Developing reliable methods to define the presence of microscopic residual disease could perhaps in the future help to identify patients suitable for treatment interruption after the achievement of clinical complete response.,27 In our patient, CTC investigation was performed through a DEPArray-based assay as previously reported implemented with antibodies directed towards epithelial-mesenchymal transition and stemness markers.,29 No actual CTC was detected in the blood of the patient with this approach, in accordance with the lasting response to therapy.In 2006, when this patient was diagnosed with metastatic breast cancer, treatment guidelines were less stringent and CDK4/6 inhibitors were not available. In this young patient we preferred to start treatment with chemotherapy because of the young age, the PgR status (0% in the bone biopsy),4 (which certainly our patient has), the long-term disease control obtained in this case with endocrine therapy alone questions the need for using CDK4/6 inhibitors in all patients with HR-positive advanced breast cancer, highlighting the need to better study predictors of treatment benefit.Our anecdotal experience highlights the existence of exceptional responders among patients with HR-positive metastatic breast cancer, who achieve clinical remission and durable disease control with endocrine therapy. Although benefit from the addition of palbociclib to endocrine therapy seems particularly relevant in patients with sensitivity to endocrine therapyMaltoni Roberta, Palleschi Michela conceived the study, wrote the article, and followed the patient. Cecconetto Lorenzo, Melegari Elisabetta, followed the patient. Rocca Andrea performed the critical revision of the article. Gallerani Giulia performed CTCs evaluation. Bravaccini Sara, Altini Mattia performed management of the data.