Interpretation of diagnostic and surveillance laboratory results and imaging in liver disease is fraught with misinterpretation and/or uncertainty. Nonalcoholic fatty liver disease (NAFLD) represents an ever-growing proportion of liver disease cases but presents unique challenges for the clinician. Given the necessity of excluding other etiologies of liver disease, NAFLD can at times represent a challenging diagnosis as non-invasive assessment and biopsy are imperfect tests with important limitations. Similarly, cautious review of laboratory reports is necessary to avoid missing abnormal pathophysiology. The presence of lab values within the standard reference range may be concerning in the setting of chronic liver disease  and conversely results flagged as abnormal may not necessarily be of great concern . This review provides a framework for the clinician to review common diagnostic challenges in NAFLD and enhance patient care. As liver disease progresses, ongoing inflammation stimulates hepatic stellate cells in the perisinusoidal space (space of Disse) to secrete excess collagen, tissue inhibitors of metalloproteinases, and matrix metalloproteinases. The sum of this activity leads to fibrosis and architectural remodeling, eventually culminating in loss of the normal lobular hepatic parenchyma in favor of discrete nodules surrounded by fibrous septa. Cirrhotic transformation occurs over many years in the majority of cases, yet its prevalence in the United States is 0.27% and is the twelfth leading cause of death overall. Historically, the primary etiologies of cirrhosis have been alcohol and chronic hepatitis C, but NAFLD/NASH has now become the second leading indication for liver transplantation. NAFLD is unique, however, as it is associated with the metabolic syndrome. Accordingly, the leading cause of death in patients with NAFLD (including cirrhosis) is cardiovascular disease (CVD), rather than liver-related complications, with degree of fibrosis on biopsy serving as an important independent predictor of mortality.Liver disease is common in the United States and has been increasing across the world, driven by the obesity epidemic and corresponding rise in nonalcoholic fatty liver disease (NAFLD). Approximately 25% of the world population has NAFLD; up to 30% of these patients have steatohepatitis on biopsy (NASH) - leading to an estimated overall population prevalence of 5%−6%To manage this increasing burden of liver disease from NAFLD, clinicians often utilize guidelines put forth by the American Association for the Study of Liver Diseases (AASLD), European Association for the Study of the Liver (EASL), Latin American Association for the Study of the Liver (ALEH), and the Asian Pacific Association for the Study of the Liver (APASL). Laboratory and imaging tests are frequently performed as part of the standard evaluation, yet much confusion exists. Liver disease, NAFLD not excepted, is fraught with interferences in homeostatic processes and there is significant heterogeneity between patients. This variance contributes to clinician discomfort (or mistaken confidence) with diagnostic algorithms and laboratory interpretation.Although a comprehensive review of the diagnostic approach to liver disease and NAFLD in particular is beyond the scope of this review, we instead aim to provide clinicians a framework to better understand common diagnostic tests. We will also specifically review situations in which labs: (1) are within the normal reference range but fail to reflect a significant underlying physiology disturbance ; and (2) are outside the normal reference range but appropriately reflect expected physiologic disturbances in liver disease .. A multitude of non-invasive tests are available in the clinician armamentarium, yet proper application and a thorough understanding of the limitations of these devices is paramount , which is problematic in fatty liver as there is a negative correlation between the amount of steatosis and speed of sound. Therefore, evaluating the speed of sound through the liver could provide an estimation of hepatic fat. Another technique described includes calculation of an attenuation coefficient . Although there are several describe methodologies in the literature, perhaps the most commercially available and validated parameter is the controlled attenuation parameter (CAP) that is typically combined with VCTE . Area under receiver operator curve values for determination of various degrees of steatosis when compared to histologic findings are good at 0.823–0.888, although not as reliable as liver biopsy or proton density fat fraction. Additionally, the accuracy of CAP in advanced fibrosis or cirrhosis is limited - thus, measurements should be interpreted cautiously in select patients,8. Several scoring systems using routinely acquired labs have been developed to estimate stage of fibrosis, which may help determine need for referral to hepatology clinic and/or liver biopsy. The most widely adopted tests include the NAFLD Fibrosis Score (NFS), Fibrosis-4 (FIB-4), and vibration-controlled transient elastography . In the absence of an established diagnosis of diabetes, calculation of the homeostatic model assessment  is required as part of the NFS for assessment of insulin resistance. Among biopsy-proved NAFLD patients, the FIB-4 carries an area under receiver operator characteristic curve of 0.86 with the NFS closely following at 0.81. Negative predictive values (NPV) for advanced fibrosis (F3-F4) are 95% and 92%, respectively, which some authors argue may allow a majority of NAFLD patients to forgo biopsy. However, recent literature suggests a more nuanced use of these scores is needed.Defining the degree of fibrosis is important in patients with NAFLD as this is the principal determinant of the risk of progression to severe liver disease and more recent evidence suggests correlation with cardiovascular risk. The utility of NFS and FIB-4 also diminishes in younger patients (age < 35), although specific cut-offs are less well-established given that the presence of advanced fibrosis is less common,11. Additionally, as age is a significant component to these non-invasive tests, many patients will have indeterminate range scores necessitating further workup. Nonetheless, these tests are easily performed in the office and have high utility as a screening mechanism in the primary care setting to triage necessity of hepatology referral and/or additional workup.The NFS and FIB-4 were originally validated in patients aged 35–65 (with the majority of patients in the 45–55-year age range) but tend to perform poorly in older patients (age > 65 years). Cutoffs of 0.675 and 2.67, respectively, for the presence of advanced fibrosis should be adjusted downward to 0.12 and 2.0 in this older cohort to improve specificity without significantly degrading sensitivityet al. determined that a kPA < 7.9 had 100% NPV for advanced fibrosis in a population of biopsy-proved NAFLD patients, with an optimal cut off of 9.9 kPA providing 95% sensitivity and 77% specificity. However, adequate performance of VCTE is limited by several factors. Hepatic congestion, such as from heart failure or the post-prandial state (hence the minimum 3 h fast required before VCTE), extrahepatic cholestasis, severe hyperbilirubinemia, operator inexperience, and obesity may preclude accurate readings. Implanted electrical devices, such as pacemakers or defibrillators, negate the ability to perform VCTE. Nonetheless, Eddowes et al. recently published reassuring data suggesting that the degree of steatosis and probe type did not affect LSM in NAFLD. This confirms prior literature validating the use of the XL probe in obese patients and/or those patients in whom reliable LSM measurements are unable to be obtained with the standard M probe.A full review of the clinical scenarios in which VCTE may be employed is beyond the scope of this manuscript, but it can be a useful tool to confirm hepatic steatosis and more importantly, estimate degree of fibrosis in NAFLD. Clinicians should also be aware that the liver stiffness measurements (LSM) provided by VCTE, measured in kilopascals (kPA), vary depending on the disease etiology. Tapper .Other available forms of elastography include proton shear wave elastography (pSWE), 2-dimensional shear wave elastography (2D-SWE), and magnetic resonance elastography (MRE). Accuracy of pSWE and 2D-SWE are similar to VCTE but limitations include obesity, a significant percentage of unreliable scans, and high interobserver variability . Although MRE is costly and requires radiologic expertise which have limited its widespread adoption, its strengths include examination of whole liver sections (thus mitigating sampling error), low technical failure rates (< 5%), and high area under receiver operator curve (> 0.90) for detection of stage 2 and above fibrosis. While massive ascites can limit signal detection, MRE is less limited by obesity as compared to VCTE, pSWE, and 2D-SWE. However, liver biopsy may ultimately be necessary in select patients. The current AASLD practice guidance suggests its use if alternative etiologies of hepatic steatosis or chronic liver disease cannot be excluded, as well as if steatohepatitis or advanced fibrosis may be present - particularly if it may influence therapeutic decisions and affect patient prognosis. Similarly, it is also common in clinical practice to utilize biopsy when there are discordant non-invasive tests, indeterminate tests, and to restage NAFLD/NASH every several years.At least two non-invasive tests are typically employed in the workup of NAFLD. Given the commonality of non-invasive biomarker scores and VCTE, a newer score called the FAST score incorporating the AST level and VCTE parameters has been designed and validated in an international cohort to predict patients at risk for NASH and progression of fibrosis. Despite these alarming numbers, NAFLD is often asymptomatic and there is substantial delay in diagnosis in non-obese patients. Interestingly, non-obese NAFLD patients typically have signs of an unhealthy metabolic profile, including dyslipidemia, insulin resistance, and hypertension - all of which are independently associated with the development of NAFLD. Additionally, there is accumulating evidence that the presence of these derangements in non-obese individuals is associated with worse liver histology and cardiovascular outcomes than obese patients without an unhealthy metabolic status.While the plurality of patients with NAFLD are obese, a substantial proportion are either non-obese (but overweight) or lean (non-overweight). A recent meta-analysis revealed that the prevalence of non-obese and lean NAFLD was 41% and 19%, respectively, within the overall NAFLD population. Perhaps more striking is that 39% of these patients had NASH and 29% had significant fibrosis.Early identification of these non-obese or lean patients is necessary and the diagnosis is often made after performance of an otherwise unrevealing workup for elevated liver enzymes or the incidental discovery of steatosis on imaging. Calculation of HOMA (see prior section) can be helpful to identify insulin resistance, as well as checking a fasting lipid panel. Non-invasive evaluation with various scoring systems, liver fibrosis markers, or VCTE can also be undertaken, as described in the prior section. However, if the etiology of steatosis and/or degree of fibrosis is unclear based on history and initial workup alone, liver biopsy may provide additional insight. Issues with adequacy of sample, patchiness of disease, and interobserver variation in steatosis and fibrosis scores are important limitations of biopsy which can affect interpretation. While HCC most commonly arises in the setting of cirrhosis from any etiology, it also may be seen in the absence of cirrhosis in some conditions. A recent meta-analysis showed that non-cirrhotic NASH patients have an increased risk of HCC compared to non-cirrhotic patients with other etiologies of chronic liver disease. However, the current AASLD guidance statement only recommends screening for HCC in patients with cirrhosis (of any etiology) and certain populations of patients with non-cirrhotic chronic hepatitis B. While this statement recognizes the increased risk of HCC in non-cirrhotic patients with NASH, the benefit and cost-effectiveness of screening remains uncertain given lack of data and the large number of patients potentially at risk.The incidence of hepatocellular carcinoma (HCC) has been increasing for the past several decades and is now the fifth most common cause of cancer worldwide and third leading cause of cancer-related death. Several strategies may be implemented to counter the diagnostic limitation of US. First, contrasted cross-sectional imaging can overcome many of the technical factors preventing adequate performance of US and has the added benefit of better characterizing hepatic lesions. Multiphase CT or MRI are the preferred modalities, with selection best determined by the clinician based on patient and local expertise factors. Second, the non-invasive GALAD scoring system has more recently been introduced as an alternative method to try to identify HCC earlier in patients with NASH, including those without cirrhosis. The score is an acronym derived from its components - gender, age, AFP-L3, AFP, and des-gamma carboxyprothrombin. In a recent hybrid study involving a retrospective case-control design which was then prospectively validated in a different cohort, the GALAD score provided high sensitivity and specificity and was able to identify patients who developed HCC over a year before diagnosis, including non-cirrhotics. While these results are promising, further validation in larger, multicenter fashion is needed.Semiannual ultrasound (US) with or without measurement of alpha fetoprotein (AFP) is recommended as the first-line screening test for HCC across all etiologies of liver disease and cirrhosis. While US is widely available and inexpensive, up to 20% of exams are considered inadequate, particularly in patients who are obese, use alcohol, or have NAFLD. However, improved diagnostic capabilities over time and confirmation of the association between metabolic risk factors and the development of NAFLD/NASH have allowed clinicians to better ascertain the etiology of cirrhosis. An early study of 70 patients with CC demonstrated that many of the cases shared risk factors of known NASH patients, including type 2 diabetes mellitus and obesity. Interestingly, these cryptogenic patients often were older females. The combination of older age, metabolic risk factors, and bland biopsies  led the authors to hypothesize that in fact a substantial portion of diagnoses may be able to be reclassified as NASH since loss of hepatic fat and steatohepatitis occur over time in NASH patients.Historically, cryptogenic cirrhosis (CC) has accounted for between 5%−30% of all cases of cirrhosis, several methodologic issues temper these findings. Further evidence that CC and NASH cirrhosis may be similar entities was shown in a study of 83 liver transplant recipients (46 with NASH and 37 with CC). Twenty patients had histologically confirmed recurrence of NASH  at a mean time of 18 months post-transplant. This group had very high rates of metabolic syndrome and insulin use before transplant, prompting the authors to advocate for targeted metabolic interventions to prevent NASH recurrence, since 30% of patients ultimately required re-transplantation.While a recent retrospective study investigating the United Network for Organ Sharing database proposed that CC may indeed be a distinct entity from NASH cirrhosis, clinicians may reasonably assume it is the underlying etiology a patient’s liver disease in the right clinical context.Given the ubiquity of serologic testing, non-invasive modalities, and performance of liver biopsy in the evaluation of liver disease and staging of hepatic fibrosis, the term CC should be used sparingly. A thorough history should be obtained from the patient, including alcohol intake (presently and over time), comorbid conditions, any prior history of excess weight/obesity (as patients with decompensated cirrhosis may have lost weight), and family history of liver disease. Although in the strict sense NAFLD may only be diagnosed when there is ≥ 5% steatosis (either by imaging or biopsy) and exclusion of significant alcohol use. Alanine aminotransferase (ALT) levels can be within normal limits anywhere along the NAFLD spectrum, ranging from simple steatosis to cirrhosis. Furthermore, patients with normal ALT and NAFLD share similar demographic and risk factors when compared to those with abnormal ALT levels. Thus, serial measurement of liver enzymes in isolation is unhelpful when following NAFLD patients longitudinally since they do not predict progression of fibrosis. However, their inclusion in validated non-invasive scoring systems is acknowledged and when combined with other clinical parameters , may help inform management and predict prognosis.Asymptomatic elevations in liver enzymes, particularly the aminotransaminases, often are a presenting feature of NAFLD/NASH. Nonetheless, normal aminotransferase levels should not reassure patients or clinicians, as they do not independently predict histologic findings or identify risk of progression to fibrosis. However, other authors argue that this is overly sensitive and would classify approximately 40% of the US population as abnormal. If not being used to identify hepatitis C-infected individuals, thresholds of 44 IU/L for men and 32 IU/L for women may be suitable alternatives. Use of these cut-offs would reduce the proportion of the US population with abnormal transaminase levels to 11%.Another important point for clinicians to note is that reference ranges for normal transaminase levels vary by laboratory. Therefore, values reflective of subtle underlying hepatic inflammation may not be automatically flagged as abnormal. Although the thresholds representing truly normal levels are subject to some debate, the most widely accepted upper limit for ALT in men is 30 IU/L and 19 IU/L for women, derived from an Italian blood donor cohort. Therefore, clinicians need to interpret liver enzymes with caution  is known to be 15% higher at baseline in African-American men and an inherent defect in enzymatic clearance which is otherwise clinically insignificant may lead to asymptomatic elevationstion see  as thereRenal dysfunction commonly accompanies liver disease, particularly in patients with cirrhosis. Current clinical estimates of renal function rely on the measurement of serum creatinine. The liver synthesizes its precursor, creatine, which is then stored in skeletal muscle and finally excreted by the kidneys as creatinine. In non-diseased individuals, there is a fairly stable total body content of creatine thus creatinine excretion approximates the glomerular filtration rate (GFR), although it does not account for the small amount of creatinine secreted by renal tubular cells.. Accordingly, a 24 h creatinine clearance is unlikely to provide any additional benefit over spot estimations based on a basic chemistry panel with the aforementioned formulas. Inulin clearance is considered the gold standard for measurement of GFR but is not routinely available in clinical practice. More recent literature proposes that measurement of serum cystatin C, a biomarker of kidney injury influenced less by extrarenal factors such as gender, age, inflammation, or muscle mass, may have utility in assessing renal function in patients with cirrhosis. A Korean study by Yoo et al. noted that the MDRD-eGFR overestimated eGFR in female patients, sarcopenic males, and patients with decompensated cirrhosis (Child-Pugh classification B or C). The same study also concluded that cystatin C-based estimation of GFR correlated better with true GFR and was superior to MDRD-eGFR in predicting overall survival and incidence of acute kidney injury. Nonetheless, one must be cautious when interpreting these results, given the MDRD was derived from an American population of Caucasian and African-American patients, who at baseline have higher muscle mass than Asian patients.Reliance on serum creatinine and traditional formulas such as the Modification of Diet in Renal Disease (MDRD), Cockcroft-Gault, and Chronic Kidney Disease Epidemiology Collaboration is highly problematic in patients with cirrhosis see . Several–43. Interestingly, this association appears independent of obesity and/or insulin resistance,43. As NAFLD has become the second leading indication for liver transplantation in the United States, the importance of recognizing sarcopenia and inaccurate estimates of renal function becomes paramount for clinicians. The current liver transplantation allocation system relies on the Model for End-Stage Liver Disease score, of which creatinine is an important component. Similarly, the guidelines to quality for simultaneous kidney transplantation also rely in part on accurate estimation of GFR. Thus, certain patients may be disadvantaged (including women and sarcopenic men) through chronic underestimation of their need for immediate organ access.While sarcopenia is well-known to occur in the setting of cirrhosis, it also has been associated with NAFLD, including NASH and advanced fibrosis,46. While hemoglobin A1c is routinely measured in these patients, its accuracy falls in patients with decompensated cirrhosis, particularly those that are severely anemic.Measurement of impaired fasting glucose and/or diabetes can be achieved through several different modalities. However, perhaps the mostly widely and easiest to use clinically is assessment of glycated hemoglobin (hemoglobin A1c) as it is less susceptible to temporary fluctuations in glycemia secondary to diet, exercise, or illness. Up to 80% of patients with chronic liver disease are known to have insulin resistance, including most patients with NAFLD. Considerred the limitations of A1c measurement, clinicians should discreetly interpret values in patients with liver disease and/or anemia to avoid potentially harmful adjustments (or lack thereof) of antihyperglycemic therapy .Despite its potential presence in NAFLD before the development of significant portal hypertension, splenomegaly, and/or cirrhosis, thrombocytopenia remains most common in these latter scenarios. Its presence should immediately prompt the clinician to perform fibrosis staging given this is known significant predictor of advanced fibrosis in NAFLD. While the pathophysiology of normal cholesterol metabolism is complex and beyond the scope of this review, it is important to understand that several of cholesterol pathways are dependent on hepatically synthesized enzymes. One of the key enzymes is lecithin-cholesterol acyltransferase (LCAT), whose activity allows esterification of free cholesterol and formation of larger (more mature) HDL particles.The liver is the principal organ involved in cholesterol homeostasis and regulates the release of very-low density lipoproteins and high-density lipoprotein (HDL), as well as the uptake of low-density lipoproteins, intermediate-density lipoproteins, chylomicron remnants, and HDL. The resulting steatosis impairs insulin signaling, contributing to insulin resistance and increasing the risk of developing type 2 diabetes mellitus. A typical lipid panel in NAFLD often demonstrates high triglycerides, low HDL, and high LDL - a pattern well known to promote cardiovascular atherosclerosis. In particular, low levels of HDL increase the risk of CVD through multiple mechanisms including but not limited to promoting efflux of cellular cholesterol, reducing inflammation, decreasing platelet aggregation, and improving glucose metabolism. While low HDL levels are common in NAFLD, CVD risk is both heightened and more severe independently of other established risk factors.In NAFLD, patients accumulate free cholesterol within the liver, stimulating Kupffer and stellate cells to induce steatohepatitis and fibrosis (lipotoxicity),59 and is a poor prognostic sign. A 2003 study by Habib et al. found that low HDL levels were predictive of 6- and 12-month mortality independent of the MELD score; levels < 30 mg/dL in particular were associated with a three-fold increase in pre-transplant mortality. Low HDL levels in patients with decompensated cirrhosis also are associated with relative adrenal insufficiency, a multifactorial endocrinologic dysfunction that may represent an impaired stress response rather than absolute glucocorticoid deficiency and independently predictive of mortality,62.Low HDL levels are also common in patients with decompensated cirrhosis of both NAFLD and non-NAFLD etiology see . This ph,64, with antinuclear antibodies (ANA) being most common (13%−21%)–66. Smooth muscle antibodies (SMA) may also be found in 3%−5% of NAFLD patients; the combination of ANA and SMA positivity is well-documented but less common. The prognostic value of autoantibodies in NAFLD remains unclear, as there is conflicting evidence in the literature regarding an association with worsened histologic features  or insulin resistance. Nonetheless, it is important to avoid diagnosing a patient prematurely with an autoimmune liver disease based solely on autoantibody positivity - patient and family history, degree of autoantibody titer, and additional serologic workup all play an important role in helping define the etiology of liver disease. Liver biopsy may be necessary if there is clinical concern for an autoimmune liver disease as the treatment for these conditions differs vastly from that of NAFLD/NASH, including potential medication-related toxicity or unnecessary immunosuppression (see The diagnosis of NAFLD (or NASH) can only truly be made after exclusion of all other etiologies of liver disease based on serologic and histologic assessment. Autoantibodies are commonly attained as part of the standard smorgasbord of labs ordered to evaluate conditions such as autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, or an overlap syndrome. However, these conditions are far rarer than NAFLD and thus without an appropriate clinical context, autoantibodies may be positive in non-autoimmune conditions. Autoantibodies are seen in 20%−35% of patients with NAFLDsion see . including excessive alcohol use, the metabolic syndrome, and NAFLD. Regarding the latter, hyperferritinemia has been associated with worse histologic activity and is an independent indicator of both steatosis and advanced fibrosis,70. The cytological changes are thought to be a result of ferritin acting as a proinflammatory cytokine with downstream activation of hepatic stellate cells and stimulation of fibrogenesis pathways in an iron-independent fashion. Interestingly, elevated ferritin levels also carry prognostic value among patients listed for transplant, including NAFLD and non-NAFLD etiologies of liver disease. In this population, a ferritin level of at least 200 μg/L has been associated with the development of portal hypertension-related decompensations, spur cell anemia, and hepatorenal syndrome, in addition to increased 6-month and 1-year mortality.Ferritin is the primary iron storage protein of the liver and may be elevated in the setting of primary or secondary states of iron overload. However, ferritin is an acute phase reactant and thus is frequently elevated in inflammatory states. Clinicians must be aware that while HH is common, there is significant phenotypic variation given incomplete penetrance. Clinically relevant iron overload occurs in patients who are homozygous for the C282Y gene; C282Y/H63D compound heterozygotes typically require a second insult  to manifest hepatic fibrosis. As metabolic risk factors often coexist in patients with elevated ferritin levels, NAFLD should be considered as either a competing or concurrent diagnosis to HH, with liver biopsy necessary to distinguish relative contributions  is unlikely but further evaluation for HH with HFE gene analysis is indicated if the transferrin saturation is ≥ 45%ions see .,75. Macrocytosis is well known to occur in the setting of alcohol abuse, however it also can result from chronic malnutrition and/or malabsorption in patients with chronic liver disease. Folic acid and vitamin B12  deficiency are common entities but diagnosis of the latter can be more challenging in the setting of liver disease. Elevated serum vitamin B12 levels are seen in 7%−18% of hospitalized patients and do not necessarily exclude underlying deficiency. An imbalance in B12 plasma binding proteins  secondary to increased synthesis or decreased clearance typically explains these high B12 levels. Specific to liver disease, elevated B12 levels result from abnormal hepatic clearance of haptocorrin and release of stored B12 from damaged hepatocytes  should be measured instead, with elevated levels suggestive of B12 deficiency .Anemia is common in liver disease, particularly in cirrhosis. A multifactorial etiology is typically present, including but not limited to hypersplenism, chronic bone marrow suppression, acute and chronic gastrointestinal bleeding, and/or hemolysis if spur cells are presentytes see ; concurr12 deficiency is likely underdiagnosed. Its prevalence in the general population rises with age, estimated at 6% in patients less than 60 years and up to 20% in patients above the age of 60. Surprisingly, autoimmune gastritis causing pernicious anemia is not frequently seen with autoimmune hepatitis. However, patients with concurrent NAFLD and diabetes may be at increased risk for B12 deficiency as long-term use of metformin can induce the condition. Although clinical guidelines around this topic are lacking, it may be prudent for clinicians to consider screening patients who have been on therapy for at least a year with measurement of MMA, as it takes between 1–5 years to deplete hepatic B12 stores.Vitamin BIn summary, this review describes challenging areas in the diagnosis and management of NAFLD. In particular, close scrutiny of laboratory results is required as labs which are “abnormally normal” and “normally abnormal” may coexist in the same patient. Clinicians who realize these intricacies will be enabled to provide excellent patient care and prevent unnecessary additional testing, procedures, or potentially harmful therapies.


Moreover, cancer treatments  may further worsen sleep problems through complex biological processes yet to be fully understood. This results in a “chicken or the egg” phenomenon, where it is unclear whether sleep disruption promotes cancer or cancer reciprocally disrupts sleep. This review will discuss existing evidence for both hypotheses and present a framework through which the interactions between sleep and cancer can be dissociated and causally investigated.Sleep is a nearly ubiquitous phenomenon across the phylogenetic tree, highlighting its essential role in ensuring fitness across evolutionary time. Consequently, chronic disruption of the duration, timing, or structure of sleep can cause widespread problems in multiple physiological systems, including those that regulate energy balance, immune function, and cognitive capacity, among others. Many, if not all these systems, become altered throughout the course of cancer initiation, growth, metastatic spread, treatment, and recurrence. Recent work has demonstrated how changes in sleep influence the development of chronic diseases, including cancer, in both humans and animal models. A common finding is that for some cancers , chronic disruption of sleep/wake states prior to disease onset is associated with an increased risk for cancer development. Additionally, sleep disruption after cancer initiation is often associated with worse outcomes. Recently, evidence suggesting that cancer itself can affect neuronal circuits controlling sleep and wakefulness has accumulated. Patients with cancer often report difficulty falling asleep, difficulty staying asleep, and severe fatigue, during and even years after treatment. In addition to the psychological stress associated with cancer, cancer itself may alter sleep homeostasis through changes to host physiology and  The importance of sleep has been recognized throughout history by the likes of the ancient Egyptians, the Greeks and the Romans-as demonstrated by the names of the gods they worshiped. Sleep is an essential component for maintaining normal physiology and re-establishing homeostasis. Sleep presents a period of vulnerability whereby the brain resides in a relative state of rest and displays reduced sensitivity to external stimuli . Despite this period of vulnerability, sleep remains a highly conserved process whereby humans (and other primates) spend a significant portion of their lives asleep-about one-third of their total lives. Additionally, the amount of time spent asleep varies greatly in mammals, varying between 3 h and more than 20 h of sleep per day . Indeed,Normal human sleep consists of two major stages- non-rapid eye movement (NREM) and rapid-eye movement (REM) sleep. Most importantly, these different sleep stages can be easily distinguished using the electroencephalogram (EEG)-measured in microvolts (μV)-to detect changes in electric charge in the brain, in the form of neuronal oscillations or “brain waves.” EEG can be used to report the collective activity of many neurons recorded by electrodes placed on the surface of the scalp. These brain waves serve various physiological functions and correlate with the different behavioral states and physiological processes that occur during different sleep stages. In addition, electromyogram (EMG) biopotential signals can be used–in tandem with EEG–as an objective measurement for sleep characterization. Interestingly, scientists have discovered that these brain waves oscillate within specific frequency bands, ranging from very slow (<0.01 Hz) to ultra-fast  oscillations . HoweverNon-rapid eye movement sleep, also known as slow wave sleep (SWS), is the first stage of sleep following the transition from wakefulness. NREM sleep constitutes about 75–80% of total time spent in sleep and is typically associated with reduced cortical neuronal activity. Hence, the most prominent brain waves in the EEG during NREM sleep are low frequency, high amplitude delta (δ) waves. These delta waves reflect synchronization between thalamocortical (TC) neurons-characterized by prolonged periods of hyperpolarization and increased membrane conductance. These changes at the cellular level are reflected at the circuit level as these periods of prolonged hyperpolarization result in a reduction in the encoding of afferent signals, thus temporarily depriving the cerebral cortex of external stimuli . In humavia brainstem GABA/glycinergic signaling  is the stage of sleep following the transition from NREM sleep. REM sleep constitutes the remaining 20–25% of total time spent in sleep and is typically associated with increased heart rate and respiration, fluctuations in brain/body temperature, increased brain activity, and active suppression of skeletal muscle activity apart from those controlling eye movement and inflation/contraction of the diaphragm. REM sleep is predominantly characterized by lower amplitude, higher frequency theta (θ) waves that reflect desynchronization of cortical neurons . Theta oignaling .Known and Probable Human Carcinogens). The synchronization of circadian rhythms is an essential component to sleep homeostasis, as the regulation of sleep/wake cycles are mainly controlled by daily circadian rhythms. In this review, we will discuss how sleep disruption may promote tumorigenesis through alterations in a variety of physiological processes such as energy balance and immunity. Reciprocally, we will discuss how cancer-mediated alterations in our physiological processes may lead to aberrant neural activity, affecting a myriad of behavioral changes including disruption of sleep/wake cycles.Our understanding of the interaction between sleep and circadian rhythms can be partially attributed to the two-process model of sleep regulation which has served as a major conceptual framework in sleep research for the last four decades. Today, we understand that sleep is regulated by a homeostatic, sleep-dependent process (Process S) that interacts with a sleep-independent circadian process (Process C) . The twoThe prevalence of sleep disorders in modern societies is increasing, affecting 20–40% of the general population , likely A growing body of research links sleep disruption and circadian misalignment to subsequent tumor initiation and growth . Given tvia immune-cell-derived cytokines) alters sleep, and reciprocally, sleep affects both the innate and adaptive arms of the immune system. Several human studies have demonstrated sleep disruption-induced elevations in circulating immune cells, including granulocytes  T cells, and B cells (2-AR) antagonist propranolol reversed the effects of sleep disruption on NK cell phenotypes. However, the causality of sleep disruption-induced alterations to the immune landscape and subsequent tumor growth remains unclear.Sleep and the immune system engage in a form of bi-directional communication, and comprise two essential components in health and disease . As we w B cells , indicat B cells . A study+ effector memory TCs (CD8 TEM), proliferating TCs , and exhausted TCs (Tex) (as a percent of CD45+). Next, JUN, FOS), IFNG and interferon-related developmental regulator 1 (IFRD1). In addition, gene ontology (GO) identified the upregulated genes across participants postSU were enriched in the AP-1 pathway, leukocyte activation, and cellular responses to stress . The most prominent downregulated genes postSU were those involved in metal ion homeostasis and detoxification. These findings indicate that sleep deprivation induces general oxidative stress and an inflammatory state in circulating immune cells. Importantly, TCs, BCs, and DCs were the cell types most strongly affected by sleep deprivation among individuals according to their upregulated genes. Within CD4+ TCs, Until recently, most studies investigating the immune landscape in the context of sleep deprivation used flow cytometric analysis with little-to-no use of high-dimensional single-cell techniques. These approaches are required to garner critical information on the molecular and cellular interactions underpinning sleep disruption associated malignancies. A study by + TCs) were altered postSU, including decreased expression of transcription factors  that normally promote differentiation and functional polarization of cytotoxic cells. In addition, sleep-deprivation upregulated levels of PFN1, a negative regulator of cytotoxic cell killing and migratory functions. Thus, the cytotoxic cells present in postSU blood show transcriptional alterations that favor an increase in an inflammatory phenotype and coincident decrease in cytotoxic activity. Lastly, Importantly, Inflammation is an evolutionarily ancient process wherein cells of the innate and adaptive arms of the immune system are activated and recruited to sites of host insult or pathogen invasion. Equally important to host defense is inflammation’s role in tissue repair and regeneration. Although vital for normal health, several decades of research have firmly implicated inflammation in the development and progression of cancer. Site specific chronic inflammation and subsequent cancer development is a common theme for many organ systems. Prominent examples highlighting this relationship include cigarette smoking and chronic viral hepatitis increasing the risk of lung cancer and liver cancer, respectively. Epidemiologic evidence also demonstrates that inhibition of inflammation with NSAIDs reduces incidence and mortality of many cancers following long term use .Substantial experimental evidence demonstrates that sleep disruption promotes inflammation in both animal models and in human studies. In one study, rats that were selectively sleep deprived of REM sleep for 72 h showed significant increases in proinflammatory cytokines compared to controls. The elevated markers include IL-1 alpha, IL-1 beta, IL-6, IL-17, TNF-alpha, corticosterone, and homocysteine. The shift to a pro-inflammatory state persisted for at least 1 week, as levels of IL-17, TNF-alpha, corticosterone, and homocysteine remained elevated despite having the opportunity for normal sleep and sleep rebound . Even juThe evidence linking sleep disruption and induction of a pro-inflammatory state is vast, however, the underlying mechanisms remain to be fully studied. Sleep induced inflammation is driven by complex neuro-immune interactions, likely mediated through neuroendocrine axes  and the autonomic nervous system, as previously discussed. As discussed above, sleep disturbance activates the HPA axis. In turn, a chronically active HPA axis can lead to glucocorticoid resistance of immune cells, wherein immune cells lose sensitivity to the anti-inflammatory effects of glucocorticoids . A naturInflammation’s role in tumor initiation is multifactorial. Sleep induced inflammation is systemic and differs from the traditional model of “site specific” chronic inflammation leading to organ-specific tumors. Cytokine and immune cell alterations following sleep disruption may create an environment favoring cancer development and progression. For example, mice that constitutively produce IL-15, a proinflammatory cytokine and growth factor, developed fatal lymphocytic leukemia . MacrophPsychological or psychosocial stress has been implicated in the etiology of several prominent diseases in humans including clinical depression, cardiovascular disease (CVD), human immunodeficiency virus (HIV/AIDS), and cancer . MoreoveSleep disruption triggers a stress response, which in turn increases concentrations of adrenal glucocorticoids and epinephrine . Research findings have firmly established that our own life-experiences, such as stressful events, have an impact on the quality, duration and physiology of sleep. Reciprocally, sleep influences stress, resulting in an overlap between stress and sleep disruption-induced physiological changes. Thus, it is difficult to disentangle the independent effects of sleep disruption alone, as sleep disruption results in emotional and physiological stress with large implications for subsequent health and disease. The connection between sleep and stress has recently been described as via the sympathetic nervous system (SNS)  ,b. The pem (SNS) . Importaem (SNS) , providiA study by In addition to the control HO neurons exert on autonomic nuclei and subsequent SNS activity in the periphery, HO neurons also modulate activity of the HPA axis. Both intracerebroventricular (icv) administration of HO and optogenetic stimulation of HO neurons results in a rapid elevation of circulating glucocorticoids , the primary humoral output of the HPA axis . In agre2-adrenergic receptors (β2-AR) on NK cells, suggesting that glucocorticoids are able to induce expression of β2-AR , stress and SNS activity are intimately linked in the periphery. Treatment of spleen-derived NK cells with the glucocorticoid receptor agonist dexamethasone upregulates expression of βof β2-AR . This reMounting evidence relates sleep loss to the development of obesity, diabetes, and other metabolic abnormalities. Metabolic abnormalities because of poor sleep are likely a risk factor for the development of many cancers. Interactions between sleep and metabolic regulation is primarily mediated through the hypothalamus. In a similar fashion to sleep, hypothalamic control over metabolism occurs through two networks that inhibit each other. Neuropeptide Y (NPY) and agouti-related protein (AgRP) neurons promote hunger and actively inhibit pro-opiomelanocortin (POMC) and amphetamine-related transcript (CART) neurons, which suppress appetite. Further, both populations of neurons are sensitive to leptin and ghrelin, which serve as major humoral cues of hunger and energy expenditure. Leptin simultaneously suppresses activity of NPY and AgRP neurons and activates POMC and CART neurons whereas ghrelin has the inverse effect in both sets of neurons .In addition to the actions of peripheral hormones on neurocircuitry, increased risk of metabolic dysfunction may be linked to sleep-induced alterations of HO neurons. Interactions between HO neurons and hypothalamic metabolic centers likely contribute to many of the metabolic abnormalities seen in those who experience chronic sleep disruption. HO neurons can sense and respond to glucose, leptin, and ghrelin, allowing for adaptive augmentation of arousal in response to changes in energy balance . FurtherLow-grade systemic inflammation due to obesity and hyperglycemia can increase the risk and progression of many cancers, including malignancy of the breast . Furthervia non-enzymatic glycation of various proteins and lipids, a process that is accelerated by hyperglycemia and inflammation. Interactions between AGEs and its receptor, receptor for advanced glycation end products (RAGEs), alters vascular homeostasis and contributes to the development and progression of cardiovascular disease . AGEs form throughout life  disease . AGE-RAG disease . AGEs hag others . Epidemig others . In summvia passive diffusion, active transport or receptor-mediated endocytosis) of essential molecules required for neural development and proper neural function. Thus, the unique structure, function, and location of the BBB enables this structure to serve as key regulator of entry into the CNS- serving a crucial role in the protection of the brain parenchyma from injury and disease.The blood brain barrier (BBB) is a complex organization of cerebral endothelial cells, pericytes, and supporting astrocytes which serves to provide a stable environment for neural function through regulation of peripheral neurotransmitter infiltration, hormones, macromolecules, and the ionic microenvironment around synapses and axons. Moreover, the BBB allows for stringent control of CNS homeostasis by monitoring and detecting circulating toxins, pathogens, and inflammation. Hence, the BBB limits paracellular permeability due to the presence of tight junctions between the continuous monolayer of endothelium in the brain vasculature. However, the presence of nutrient transporters  and receptors  on endothelial cells lining the cerebral vasculature enables the delivery  ] which are less active at night , thus increasing permeability of the brain overnight. The presence and activity of Pgp on brain capillary endothelial cells serves as an efflux pump to expel substrates back into the circulation after they initially diffuse into the endothelial cells, restricting entry into the brain parenchyma. Additional studies have demonstrated the role of sleep loss in impairment of BBB function and subsequent increase in BBB permeability to proinflammatory cytokines  and immune cells  . In addiansport) . This isne cells . A studyIn alignment with these findings, previous studies in rats have demonstrated that co-culture of endothelial cells and pericytes are more effective in increasing transendothelial electrical resistance (TEER) and lowering permeability to both low and large molecular weight tracers as compared to monocultures of endothelial cells alone . In addiMertk expression- a “wake” gene upregulated in both spontaneous wake and sleep deprivation. In addition, Glial cells are present in both the central and peripheral nervous systems and serve many vital functions  and are essential regulators in the formation, maintenance, and function of synapses. The major type of glial cells in the central nervous system  are astrocytes, oligodendrocytes, microglia, and ependymal cells–each of which contain their respective function and molecular/cellular characteristics. Neurons and glial cells occupy a comparable amount of space in nervous tissue; however, the number of glial cells outnumber that of neurons. Thus, a comprehensive review of the effects of sleep disruption on tumorigenesis–and reciprocally, the effects of peripheral tumors in promoting sleep disruption–must encompass the potential contributions of glial cells. Recently, a role of glial cells in the regulation of sleep-wake cycles is emerging, highlighting the dynamic interactions present within neural circuits that influence sleep in both health and disease . For exaPoor sleep experienced by cancer patients and survivors could be attributed to the presence of one or more underlying sleep disorders. Thus, one of the most prominent concerns in cancer patients is the onset of sleep disorders including difficulty falling asleep, problems maintaining sleep, poor sleep efficiency and early awakening. Early immunologists treated the nervous and immune systems as largely separate entities. However, today there is consensus that there is bi-directional communication between the nervous and immune systems in both health and disease states. To date, there are several pathways that have been proposed in the field of neuroimmunology as being important for the communication between the immune system and central nervous system: the neural route , BBB active transport of cytokines and secretions from BBB cells, passive diffusion at circumventricular organs which lack a BBB , infiltration of peripheral immune cells, as well as interactions with meningeal lymphatics . Thus, pvia humoral and neural routes) and subsequently alter neural activity  cognitive capacity. The nervous system and peripheral tumors  engage in bidirectional communication as well. A growing body of literature illuminates the role of nerves in cancer initiation, progression, and subsequent metastasis through direct interactions with cancer cells or through interactions with stromal cells in the TME . Just asactivity . Consequactivity .Stat3). In addition, brain and muscle activity were detected using EEG and EMG, respectively, to assess changes to sleep-wake states throughout cancer progression. Interestingly, the presence of the peripheral tumor was found to disrupt sleep-wake states in the later course of tumor progression as evidenced by reduced time spent awake and an increase and fragmentation of NREM but not REM sleep. These observed sleep-wake aberrations were mainly attributed to enhanced activity of HO neurons as evidenced by increased cFos immunoreactivity during the active phase in tumor-bearing mice. Importantly, the altered sleep patterns were not due to altered immune activation in the brain, as there were no indications of increased pro-inflammatory cytokines  in several brain regions. via the neurotoxin 6-OHDA) attenuated metabolic abnormalities in tumor-bearing mice as evidenced by restoration of blood glucose concentrations and normalization of several hepatic genes involved in gluconeogenesis/glycolysis . These findings provide mechanistic insight into tumor-driven alterations in sleep-wake neurocircuitry that may be coupled to changes in metabolism or immune signaling in the periphery. These findings warrant further discussion on reprogramming the use of current clinically approved hypocretin receptor antagonists  for improving metabolic and sleep aberrations in cancer patients  of the spinal cord or along cranial nerves. Sensory neurons are subdivided into visceral and somatic sensory neurons, which transmit sensory information primarily from internal organs and skin and skeletal muscles, respectively. In addition, sensory neurons contain different receptors for different stimuli  which in turn allow for the perception of various sensations including pain, temperature, and touch.via ascending pathways, conveyed to various brain regions to elicit pain sensations. Nociceptors are capable of detecting different forms of noxious stimuli  that are secreted by cancer cells and other components of the TME  . Since tvia vagal input and likely enter the brain parenchyma via aforementioned mechanisms  resulting in aberrant neuronal activity.Interestingly, several studies highlight a contribution of the vagus nerve in the sleep-promoting effects of IL1-B. A study by Sleep is regulated by both humoral and neuronal mechanisms that are dependent on each other. Several studies have demonstrated that proinflammatory cytokines  have a somnogenic effect, increasing both sleep  and lethargy following peripheral immune activation . Interesvia vagal afferents  and in vivo as analyzed by human CNS postmortem tissues from individuals with multiple sclerosis, a disease with well-characterized BBB disruption. Kebir et al. also demonstrated that the binding of both IL-17 and IL-22, two identified cytokine products of TH17 lymphocytes, to their receptors  on human brain endothelium was critical for the increased permeability of the BBB. IL-17 consists of a family of proinflammatory cytokines secreted primarily by activated T-helper type 17 (TH17) lymphocytes. IL-17 plays an important role in the homeostasis of tissues in health but also contributes to autoimmunity, chronic inflammation and invasion in various inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases and type I diabetes , and relaxation techniques. CBTI is an evidence-based, structured program that is used to combat insomnia in the general population. Recent trials have found that CBT-I can significantly improve sleep measures in breast cancer survivors and patients undergoing treatment . ResultsIn vivo studies have demonstrated that melatonin treatment reduced tumor size and cell proliferation, as well as a decrease in VEGF receptor 2 density, in mice with breast cancer xenografts  should be leveraged to dissect the neural circuits disrupted by cancer, leading to a more complete understanding of the pathophysiology of malignancy. Uncovering the neural correlates of cancer-related sleep disruption has the potential to inform novel treatment approaches and screening tools that will improve patient care and oncology outcomes. Despite recent progress, many questions remain regarding sleep in the context of cancer.A potential focus for preclinical discovery will be to localize specific sleep-associated brain nuclei deregulated by cancer and then employ targeted stimulation or inhibition of these circuits to see what their role is in the development of cancer-associated sleep disruption. Advances in neuroimaging, such as serial two photon tomography (STPT), are already allowing researchers to examine how an entire brain responds to selective perturbations  . UnbiaseA detailed understanding of how cytokines and metabolic factors affect the central nervous system will become increasingly more important as future cancer treatments targeting or harnessing the immune system become more widely used. Immune checkpoint inhibitors, such as antibodies targeting PD-1/PD1 or CLTA-4, and cellular therapy utilizing chimeric antigen receptor (CAR) T cells are all likely to alter the cancer-induced cytokine and metabolic milieu. Developing a neuroimmune effector map now will translate into the ability to predict the effects targeted immunotherapies may have on the CNS.in vivo. Finally, we need additional input from oncologists, as not all cancers influence sleep in the same way, and the amelioration of these problems will likely require close collaboration with those who know the details of specific cancer-secreted molecules, propensity for neural invasion, and treatment resistance.Computational approaches will be key in handling the large amounts of data generated by EEG/polysomnography and pulling out important cancer-related changes in brain function. These include machine learning/artificial intelligence techniques, which can (in certain situations) identify small but important changes in data structure that may be missed by classic sleep scoring techniques. This will promote a research cycle where computational approaches allow us to generate hypotheses from large datasets that can then be tested and refined AB prepared figures and figure legends. JB supervised the work. All authors designed, wrote, and edited 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Structure determination of membrane proteins has been a long-standing challenge to understand the molecular basis of life processes. Detergents are widely used to study the structure and function of membrane proteins by various experimental methods, and the application of membrane mimetics is also a prevalent trend in the field of cryo-EM analysis. This review focuses on the widely-used detergents and corresponding properties and structures, and also discusses the growing interests in membrane mimetic systems used in cryo-EM studies, providing insights into the role of detergent alternatives in structure determination. Integral membrane proteins play critical roles in many physiological processes including metabolism, signal transduction, and energy utilizationDetergents are indispensable when working with integral membrane proteins. By nature of their amphiphilic character, detergents can partition into biological membranes, extract proteins, and maintain protein solubility in solution. The usage of detergents in membrane protein crystallization has been extensively reviewed over the last few decades –8 , whiThis review first briefly discusses the basic properties of detergent and compare widely-used detergents between different experimental methods in recent years. This review then provides detailed insights into various effect of the detergent micelle on structure determination and corresponding structures, particularly in cryo-EM analysis. Here I further discuss the growing interests in membrane mimetic systems which provide a more native environment for integral membrane proteins, and focus particularly on the role of detergents alternative for structural and functional studies.Detergents play an essential role in the extraction, purification, manipulation, and structure determination of membrane proteins. The amphiphilic characteristic of detergents allows them to interact with hydrophobic membrane proteins and to provide a native bilayer environment; it facilitates the extraction of proteins from membranes by disrupting the bilayer structure, then maintains the proteins in a soluble formAccording to the property of hydrophilic head, detergents can be categorized into three groups: ionic, non-ionic, and zwitterionic detergents. Detergents with small, charged head groups and relatively short alkyl chains are generally much more denaturing to membrane protein structuresApart from these, different modes of solubilization selection appear when classifying structures by protein types and structural determination methodsGiven that the optimal extraction conditions may not be the best conditions for further characterizationIn addition, some detergents are often added to protein to improve grid preparation in cryo-EM samples. For example, zwitterionic detergent CHAPSO significantly broadens the particle orientation distributions for a number of bacterial transcription complexes  35,3636 , whicMicellization is a critical phenomenon when considering detergent application. The concentration of detergent above which micelles form and all additional detergents added to the system go to micelles is described as CMC. Detergents with lower CMC are desirable in cryo-EM studies, such as GDN and LMNG, for which can minimize the detergent concentration while keep the protein soluble. Here I consider the effect of micelle on structure determination, particularly in the field of single particle cryo-EM.In most cases, detergent levels in membrane protein samples are typically kept above the CMC to allow the hydrophobic parts of the protein incorporated into micellesOn the other hand, a clearly delineated micelle may aid in the single particle classification and alignment. Micelle size varies with different detergents and concentrations, even not all micelles appear as spherical. For example, LMNG appears as worm-like filament in negative-stain imagesThe application of detergents in cryo-EM studies often causes complications such as increasing thickness of vitrified ice, reducing contrast, making background noise and particle distribution problems. To date, several novel membrane mimetic systems without the presence of detergents have been introduced to study integral membrane proteins , includiIn the past few years, the most extensively used detergents alternatives are nanodiscs and amphipolsNanodiscs consist of discoidal phospholipid bilayers stabilized by two belt-like membrane scaffold proteins (MSPs) derived from apolipoprotein A-1 B 65,6,665,66Reconstitution of most membrane mimetic systems requires solubilization and purification of membrane proteins in detergents at first. By contrast, SMA copolymers can directly insert into the bilayer and extract integral membrane proteins within natural lipids D 69,7,769,70All the solubilization systems mentioned above are proved to be reasonable and practical, which can be successfully applied to structure determination of membrane proteins. Here I introduce an example of pannexin1, illustrating one membrane protein structure in parallel, to confirm the consistency of different detergents and membrane mimetic systems. Pannexin1 (PANX1) is an ATP-permeable channel with crucial roles in physiological functions such as regulation of blood pressureWith technical development and facility renewal, single-particle cryo-EM has strongly affected the usage of detergents and membrane mimetics, resulting in a spurt of membrane protein structure determined. Conventionally used detergents, despite its deficiencies, is undeniably still a major force for the purpose of high-resolution structure determination. Therefore, the choice of detergents and alternatives should be considered comprehensively according to the experimental methods, protein type, and research target. This review also summarized representative conditions used for membrane protein structure determination by cryo-EM mainly mentioned in this review , which i


Genes are transcribed to produce pre-mRNAs, which are then spliced to create the mature mRNAs translated into protein. In recent years, improved deep sequencing technologies have shown greater than 90% of human pre-mRNAs undergo alternative splicing, thereby amplifying the potential protein products from each gene . AlternaMany growth factor and hormone receptors and signaling molecules implicated in cancer have natural splice variants, some of which have been shown to act as dominant negatives. We hypothesized that by altering splicing to decrease growth-promoting and/or increase expression of dominant negative varieties we could eliminate abnormal dependence on growth factors, decrease metastatic potential, and promote cancer cell death.By binding to specific intronic or exonic regions or intron-exon junctions, splice modulating oligomers, which are cDNA sequences, can alter the outcome of splicing . To our knowledge, no one had previously tapped the potential of splice modulating oligomers to increase the relative activity of natural dominant negatives in order to combat disease. Where splice modulating oligomers had begun to be explored as therapeutics was for diseases that result from splicing errors and the production of a non-functional protein , 5.Dominant negative receptors may inhibit signaling from the growth-promoting form of the receptor in a variety of ways. In the simplest situation, a dominant negative receptor binds ligand and therefore reduces availability to the growth-promoting receptor. In other instances, the dominant negative receptor may generate an alternate intracellular signal e.g., 6–9]. Such amplification of the effect of dominant negative receptors through a signaling cascade makes an increase in their relative expression all the more effective. Importantly and additionally, the signals generated can promote differentiation and/or apoptotic cell death –9. Such , therebyAs our proof of principle, we have applied splice modulating DNA oligomers to knockdown the proliferation-promoting and antiapoptotic long isoform of the prolactin receptor (LFPRLR), while still allowing signaling from the short, differentiation-promoting and pro-apoptotic forms. Although prolactin signaling is important to initiation and progression of prostate, ovarian, breast and blood cancers, our initial focus has been on breast cancer. Thus, we have tested the efficacy of splice modulating oligomers against highly aggressive, metastatic breast cancers in both syngeneic and human xenograft situations , 11. Knovice versa (Accumulation of tumor T regulatory cells (Tregs) is associated with a worse prognosis in most cancer types. Orthotopic, syngeneic tumors of mouse triple negative breast cancer cells are so attractive to Tregs that one can demonstrate Treg depletion from the circulation . Systemice versa . Moreove in vivo where local cytokines/chemokines can influence the outcome.Therapeutics targeting Tregs are proving to be effective treatments for many cancers. However, because they target all Tregs, current immunotherapeutics can result in development of a variety of inflammatory disorders and autoimmune diseases [discussed in 11]. Even though all Tregs constitutively express LFPRLRs, the LFPRLRs in Tregs are normally resistant to knockdown, most likely because a splicing regulator occupies the target site of the splice modulating oligomer. Therefore, there was no negative effect on peripheral Treg function [Adding to their attractiveness as therapeutics, splice modulating oligomers can be combined (or sequenced) to target several different transcripts, potentially increasing the impact of treatment, and likely making it more difficult for tumor cells to become resistant. In addition, as morpholino DNA oligomers they are more stable than short interfering or antisense RNAs and therefore do not require carrier technologies. They can be derivatized in a number of ways to promote cell penetration; in our studies, we used octaguanidine dendrimer derivatization and have shown very good knockdown in multiple tissues with no discernible toxicity at effective anti-tumor doses , 11. In


In recent years an expanding collection of heart-secreted signaling proteins have been discovered that play cellular communication roles in diverse pathophysiological processes. This minireview briefly discusses current evidence for the roles of cardiokines in systemic regulation of aging and age-associated diseases. An analysis of human transcriptome and secretome data suggests the possibility that many other cardiokines remain to be discovered that may function in long-range physiological regulations. We discuss the ongoing challenges and emerging technologies for elucidating the identity and function of cardiokines in endocrine regulations. The heart is one of the most important organs in terms of age-associated diseases and public health burden in an aging world. An emerging paradigm has focused on how circulatory factors can both contribute to and protect the heart from cardiac pathophysiology in aging and age-related diseases, including circulating growth differentiation factor 8/myostatin (GDF8/MSTN) , fibroblin vitro and in vivo. A large complement of these heart secreted proteins has been identified including prominently autocrine and paracrine growth factors, pro-angiogenesis factors, and extracellular matrix remodelers, which have been reviewed comprehensively elsewhere. For the purpose of aging research, here we propose a more limited definition of a bona fide cardiokine. In our view, a bona fide cardiokine represents a peptide or protein that: 1) is detectable in systemic circulation and has the potential to signal to distal tissues in an endocrine rather than autocrine or paracrine fashion; 2) shows some degree of tissue-biased expression, where an appreciable fraction of a circulating pool is due to secretion from the heart, either in the baseline or in a given diseased condition; and 3) exists as a free extracellular protein or peptide rather than as a cargo of extracellular vesicles, and acts directly on a ligand receptor. This definition of cardiokines is therefore distinguished from the cardiac secretome at large, which includes many local interstitial and matricellular proteins that function in various paracrine and autocrine contexts including a large number of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that function in cardiac extracellular matrix remodeling during aging  family protein that is translated as a 41 kDa pre-prohormone. The signal peptide and prodomains are then cleaved to form a dimer of the 12.5 kDa mature circulating peptide. Both GDF8/MSTN and the closely related growth differentiation factor 11 (GDF11) have been considered putative anti-aging proteins whose decline in age is positively correlated with cardiovascular events and death . GDF8/MS failure , thus suGdf15, suggesting GDF15 functions as a heart-to-liver endocrine signal that inhibits body growth in the mouse  receptor . GDF15 lhe mouse .Fgf21 increased circulating FGF21 levels in the mouse, and at the same time led to a moderate decrease in body mass, reduction of lean mass, glucose level, and increase in body fat mass ; as well as 168 such transcripts in the left ventricle (141 increasing with age and 27 decreasing) . RelatinBesides tissue expression enrichment or specificity, cardiokine-coding transcripts can show tissue bias in age-associated expression changes. These tissue-biased expression changes may reflect pathophysiological cues and signals that are masked in plasma protein level measurements and suggest the possibility of increasing cardiac contributions to the circulating pool in some scenarios. To illustrate, GDF15 has broadly increased expression with age in both cardiac tissues as well as skeletal muscle and adipose samples , whereasGiven the prevalence of secretome coding transcripts with age-expression trends in cardiac tissues, one might wonder whether more cardiokines stand to be discovered that function in aging. We believe several technical challenges currently exist with regard to the identification of bona fide cardiokines and their role in aging and age-associated diseases.The discovery of unknown circulatory proteins in the plasma remains difficult. Blood can contain potentially every protein in the human proteome and has a steep dynamic range of concentration which makes it challenging to identify by mass spectrometry. Affinity-based assays have high sensitivity, but their target specificity has been called into question . Circulain situ. Which tissue dominates as the source of the total circulating pool in the plasma may be dependent on pathophysiological conditions, and a challenge pertains whether the sampled proteins form a single pool or whether the cardiac-released form has distinct function, which may be due to spatiotemporal, concentration-dependent, isoform and proteoform differences. These complications may be partially resolvable by comparing cardiac coronary blood to systemic circulation.The tissues-of-origin of a protein are difficult to deconvolve from systemic circulation. Plasma is a mixed pool of proteins from different tissues. With possibly the only exceptions of ANP and BNP, cardiac secreted proteins are also released by multiple other tissues, where they likely have different, pleiotropic functions Similarly, it remains a challenge to unravel the target cell and tissue types of signals and associate function. The majority of studies on cardiac secreted proteins focus on characterizing their autocrine/paracrine effects on cardiac physiology and disease responses, hence an age-associated effect in distal tissues may be missed, and this generally contributes to the difficulty of assigning systemic/endocrine physiological functions. Mouse models of cardiac-specific deletion of GDF11 and transgenic expression of GDF15 and MDK exist, but to our knowledge existing reports have only compared their cardiac phenotypes. Large-scale physiological phenotyping comparisons, such as performed on global vs. cardiac-specific GDF8/MSTN knockout mouse model , are neeLastly, cardiokines are often triggered by myocardial infarct, heart failure, fibrosis, and other age-associated disease conditions . The obsComputational approaches using correlation networks from large-scale sequencing data have been employed to find tissue-specific proteins as well as proteins potentially involved in cross-cell communication. Co-expression analysis is frequently used to identify functionally related genes, based on the assumption that gene pathways may be co-regulated by an unobserved genetic architecture . This apin vitro. The secretome of the resulting cells can then be collected and analyzed using multiple types of omics techniques in vitro to discover and confirm secreted proteins, (Human induced pluripotent stem cells (iPSCs) provide an experimental tool to identify the secretion and downstream signaling function of cardiokines in cells and tissues differentiated from human iPSC lines roteins, , or convroteins, . This boroteins, . A similMore importantly, multiple cell types from different organs can be simultaneously differentiated that originate from identical iPSC lines and thus carry identical genetic backgrounds, which removes the confounding factors of genetic variance. Engineered “body-on-a-chip” systems containing both iPSC-derived cardiomyocytes and another cell type such as hepatocytes connected by microfluidic devices, for example, have garnered considerable enthusiasm for their potential uses in drug screening and disease modeling . Many coThere is increasing interest in the endocrine functions of the heart and its intersection with human aging and age-associated diseases. Many cardiac secreted proteins are now identified, although thus far few have been established to exist in systemic circulation beyond the cardiac interstitial space, and fewer still have established endocrine signaling functions to distal tissues. Nevertheless, an analysis of human transcriptomics and proteomics data suggests more cardiokines with age-associated expression are likely to be discovered. We believe there is both an opportunity and need to focus on finding long-distance circulatory signals originating from the heart. Future studies may achieve this goal using next-generation stem cell models, multi-omics, and bioinformatics techniques.


Advances in tissue engineering have progressed to potentially offer a solution to temporomandibular joint disc (TMJ) disorders not amenable to conservative therapies. Conclusive treatment options for patients with end-stage disc disorders requires discectomy and reconstruction of the articular disc with various materials. Tissue engineering TMJ disc is a promising alternative to the limited and sometimes inadequate clinical options in the management of such disorders. However, tissue engineering is far from completion for the TMJ disc regeneration. This review briefly discusses the properties of native disc, the mechanism by which TMJ disorders manifest, and how a tissue engineered disc could assuage the problems inherent in the management of such disorders. Furthermore, the review addresses and provides updates to relevant themes of tissue engineering in regards to the TMJ disc, namely, the scaffolds, cells and biomarkers, hurdles in tissue engineering of the disc, and its application in translation to the clinical practice and future directions. The temporomandibular joint (TMJ) is a synovial joint between the mandibular condyle and the glenoid fossa of the cranium. The temporomandibular disc is a fibrocartilaginous tissue with a biconcave shape that allows it to fit the bony anatomy and divide the joint into a superior and inferior compartment . It is vIn a normally functioning TMJ, the continuous wear and tear of joint surfaces and the disc are repaired by the reparative cells present in the synovial fluids and the joint surfaces, but this intricate and delicate balance is oftentimes disrupted by unusual loading due to micro or microtrauma to the joint, the result of which is displacement, elongation, and fragmentation of the disc which bears the brunt of initial forces before it gives way and the joint surfaces are exposed to trauma . SymptomTissue engineering (TE) in its essence tries to solve the inherent problems associated with autogenous grafts, namely, donor site morbidity, the inability of autografts to be a replica of the desired tissue anatomically, morphologically and histologically, and lack of mechanical properties required to rehabilitate the organ to its prediseased functional state , 19. In We searched PubMed and Google Scholar with the following search algorithm: “tissue engineering” AND “temporomandibular disc. Only recent articles published after 2010 were considered to present recent advances in the field. This is a scoping review to update the scientific advancement on this topic; hence, protocol registration was not performed.Multiple reviews on the topic were analyzed, each reporting on different themes concerning TMJ disc tissue engineering. Recent in vitro and preclinical animal studies were analyzed which presented outcome data of in vivo TMJ disc implants generated with various materials as scaffolds, cell sources, and signaling biomarkers. These findings are presented according to these themes: scaffolds, cell sources and biomarkers, hurdles in tissue engineering of TMJ disc, and future directions.Scaffolds are natural, synthetic, or combination structures that act as a template and carrier of cells and biosignals for the neoformation of a tissue , 21. An The first used natural scaffolds were collagen fibrous mesh which showed promising results with spontaneous extracellular matrix (ECM) and cartilage formation but was limited by foreign body reaction around the implant. Fibrin, agar, and alginate gels are used as hydrogel which exhibits excellent water retention capacity and encloses seeded cells uniformly, but lack the mechanical strength, shrink in volume, and rapidly degrade; thus, they are disqualified as an ideal scaffold material for TMJ disc TE –25. Autoβ1 results in improved mechanical properties and higher collagen and ECM deposition compared with the PGA control [Synthetic materials provide freedom and flexibility, as they can be modified in most parameters, processing capacity, mechanical stability, biocompatibility, biodegradability, pore size, and geometry , 28. An  control . PLGA (p control .2) allowed cell adhesion and upregulation of fibrocartilage formation. Additionally, the composite scaffolds showed similar storage modulus and elastic response comparable to the native TMJ disc.A way forward to compensate for the limitations of individual scaffolds is to fabricate composite scaffolds, utilizing multiple materials, each with different desired properties, to end up with an ideal material close to the native disc . A compoβ3 can be 3D printed mimicking spatially distributed collagen of TMJ disc. Histologically accurate tissue similar to native TMJ disc with a full recovery of the perforated disc was observed in vivo. A study used PCL, photopolymerized hydrogel polyethylene glycol diacrylate (PEGDA), and a combination of the two materials. The combination material showed the best results, as the slow degradation of PCL was congruent with the slow growth rate of the TMJ disc. Moreover, PCL with its relative rigidity provided adequate mechanical properties which could be advantageous during surgery, whereas PEGDA hydrogel promoted cellular adhesion and lubrication. Thus, a reduction of joint friction and the distribution of the functional load allowed by PEGDA and scaffold geometry and mechanical properties of PC demonstrated that the combination of materials could lead to a desired progress in the TMJ disc TE.The combination of synthetic polymers, PLGA, and polycaprolactone (PCL) seeded with recombinant human bone morphogenetic protein 2 (rhBMP-2), connective tissue growth factor, and TGF-3D printing of scaffolds can achieve high precision and accuracy, with the ability to fabricate complex geometric shapes with close to native spatial cell distribution and mimic the native ECM , 33. A lIn a study using nanoassembly and nanocoating technologies, titanium dioxide surface modification was implemented with TMJ disc cells which showed a proportional increment in cell proliferation and extracellular matrix (ECM) deposition with increasing thickness of nanofilms  layered To bypass the complications of scaffold-based TE, the scaffold-free self-assembling process has been reported to generate a functional disc resembling the native disc which is mechanically robust . HoweverIn a randomized controlled preclinical trial on interpositional TMJ discs in the black merino sheep model (TEMPOJIMS) , the autThe success of TMJ-related tissue engineering is dependent upon selection of cell source that are utilized for seeding the scaffolds. The primary cell source in TMJ tissue engineering include autologous TMJ disc cells, articular chondrocytes, costal chondrocytes, and allogenic cells. The native TMJ disc cells have been of great interest of many researchers; however, the challenges associated with healthy tissue such as donor site morbidity and degenerative changes have also been reported . Due to Stem cells  have been successfully used in TMJ tissue engineering. The mesenchymal stem cells (MSCs) have self-renewal property and have capacity for differentiation into various cell lineages. The MSCs have been explored to a great extent for the treatment of medical problems due to their potential role in tissue repair and reduction of inflammation . They haNotably, MSCs derived from synovial fluid and synovium have stemness trilineage differentiation, self-renewal capacity, and immunosuppressive properties . Both syβ1 (TGF-β1), and insulin like growth factor (IGF) [The use of type I collagen matrix isolated from rabbit tendon favored the regeneration of TMJ articular disc in rabbit . In anotor (IGF) .It has been well documented that laser treatment modulates cellular properties. Photobiomodulation have been used to decrease inflammation and promote wound healing. Photobiomodulation with low-level laser therapy has shown to reduce pain sensation in rats . StudiesIn addition, various growth factors such as bone morphogenetic protein 2 (BMP-2), connective tissue growth factor (CTGF), and platelet-rich plasma (PRP), epidermal growth factor, interleukin 1, and tumor necrosis factor alpha used alone or in combination with cells and/or scaffolds have a role in proliferation and production of collagen and glycosaminoglycans (GAG), and hence, they have been used to regenerate TMJ discal tissues too . Among tα/Nf-κB promoted fibrocartilage stem cell's chondrogenic potential [β1 and IGF-1 induced increase in type 1 collagen and GAG synthesis and cells proliferation [Apart from this, other factors such as platelet derived growth factor, epidermal growth factor, interleukin-1, and tumor necrosis factor alpha also have role in proliferation, production of collagen, and glycosaminoglycans (GAG) . In a reotential . Kang etferation . In a stferation .  FigureScaffolding material for TMJ disc TE remains a challenge because these materials exhibit differential contraction, insufficient mechanical strengths for load-bearing, degradation of the products, stress-shielding, and material-induced immunologic responses . Novel tIn a recent study on minipigs, TE discal implants were compared to empty controls. The tissue engineered discal implants restored “disc integrity by inducing 4.4 times more complete defect closure, formed 3.4-fold stiffer repair tissue, and promoted 3.2-fold stiffer intralaminar fusion. The osteoarthritis score (indicative of degenerative changes) of the untreated group was 3.0-fold of the implant-treated group.” These encouraging findings support that theoretically TE TMJ discs can one day supplant current grafts and alloplasts as the most convenient and effective treatments . BioprinAn ideal TMJ disc implant should have a few desirable properties; first, it should be able to withstand masticatory functional loads for a considerable long period or loading cycles; second, it should have a similar histological, cellular, and extracellular matrix profile, should have high biocompatibility and feasible to produce, and have familiar mechanical properties of the native disc. Current limitations of tissue engineering strategies for implantable TMJ disc analogue are not near completion; nevertheless, they represent a promising future in the disc replacement strategies for the clinicians. Safety issues related to immunologic response towards the brain tissue is critical in formulating indications for the application of TE TMJ disc in clinical practice.


Despite significant advances in developing drugs to treat brain tumours, achieving therapeutic concentrations of the drug at the tumour site remains a major challenge due to the presence of the blood–brain barrier (BBB). Several strategies have evolved to enhance brain delivery of chemotherapeutic agents to treat tumours; however, most approaches have several limitations which hinder their clinical utility. Promising studies indicate that ultrasound can penetrate the skull to target specific brain regions and transiently open the BBB, safely and reversibly, with a high degree of spatial and temporal specificity. In this review, we initially describe the basics of therapeutic ultrasound, then detail ultrasound-based drug delivery strategies to the brain and the mechanisms by which ultrasound can improve brain tumour therapy. We review pre-clinical and clinical findings from ultrasound-mediated BBB opening and drug delivery studies and outline current therapeutic ultrasound devices and technologies designed for this purpose. Despite the growing body of literature in the development of novel and promising therapies, treatment of primary and metastatic brain tumours remains a major challenge worldwide ,2. In paVascular permeability within a brain tumour  is heterogeneous and is on average greater than the BBB. This results in penetration of the anti-cancer drug through the BTB and into tumour tissue. However, the increase in drug permeability is not enough to achieve an effective tumour therapy ,5,6,7. SOver the past few decades, nanomedicine has gained significant interest in the field of controlled and targeted drug delivery systems (DDS) . Nano/miFocused ultrasound (FUS) is currently the only available technique that can induce selective and localized opening of the BBB non-invasively in humans. The discovery of FUS-induced BBB opening has remarkably facilitated brain drug delivery , particuHere, we will provide a comprehensive review of the application of ultrasound in controlled drug delivery, from the basics of ultrasound and sono-responsive carriers to the clinical application of sophisticated ultrasound transducers for treating brain tumors. We will initially review the challenges in treating brain tumours, and strategies used to bypass the BBB/BTB to enhance drug delivery. Sono-responsive drug delivery, and therapeutic ultrasound devices developed for this purpose, will then be reviewed in detail. Finally, pre-clinical models and clinical studies using ultrasound-mediated BBB opening for the treatment of brain cancers will be discussed.Malignant gliomas, of which glioblastoma (GBM) is the most common primary brain cancer affecting adults, cause one of the highest levels of morbidity and mortality among cancer patients . This moMultiple mechanisms contribute to treatment inefficacy. Firstly, complete surgical resection of brain cancers in many cases is nearly impossible due to their anatomical location and infiltrative nature. Secondly, the use of radiotherapy is often restricted, due to its potential deleterious effect on surrounding normal brain tissue. Thirdly, aggressive brain cancer cells show significant resistance to chemotherapy. Lastly, the BBB or BTB restricts the accessibility of malignant tissues to most chemotherapeutic drugs, thereby limiting the effective tissue concentration of agents to which malignant tissue might otherwise be sensitive .The brain tissue landscape and its specific microenvironment is distinct to many other tissues ,25, mostThe BTB is structurally highly heterogeneous and tortuous, with non-uniform and irregular permeability and active efflux of molecules, in comparison to normal BBB . For per®) for the treatment of Kaposi’s sarcoma in 1995  an an117] a2 at 2.6 MHz) of Chinese hamster lung fibroblast cells did not significantly impact cell viability. However, when the sonication was combined with doxorubicin treatment, it resulted in a significant enhancement in the cytotoxic activity of doxorubicin ..211].Thermal ablation (the process of removing tissue using the heat generated by HIFU) by transcranial non-invasive FUS is potentially the most straightforward approach for brain tumour therapy. However, achieving adequate tumour necrosis and minimal off-target effects remains the major challenge ,252. LowTwo clinical trial studies  have been performed in adults with recurrent GBM treated with intravenous carboplatin, using the pulsed ultrasound systems SonoCloud-1 or SonoCloud-9, respectively. These trials have indicated that repeated transient BBB opening, in combination with systemic MB injection, is safe and has the potential to optimize chemotherapy delivery such as carboplatin in the brain ,247. TheAnother clinical trial of low-intensity FUS-BBB opening with systemically administered liposomal doxorubicin or temozolomide in patients with malignant brain tumours has been reported (NCT02343991) . The BBBAs mentioned above, another application of ultrasound-mediated BBB opening could be for treating CNS neurological disorders treatment such as amyotrophic lateral sclerosis (ALS), Parkinson’s and Alzheimer’s disease  in five patients with early-to-moderate Alzheimer’s disease demonstrated for the first time that the BBB within the target volume was safely, reversibly, and repeatedly opened. No clinically severe adverse events nor clinically significant worsening in cognitive performance 3 months after opening the BBB were observed . UltrasoDespite the meteoric developments in brain tumour drug delivery, overcoming BBB and BTB is a major challenge which limits the efficacy of these treatments. In addition, most approaches result in new local and systemic side effects. Among all of these approaches, ultrasound-mediated drug delivery holds great potential for designing non-invasive, targeted pharmacologic neuro-interventions. However, despite the promising pre-clinical and clinical findings, there are several limitations with currently available ultrasound-mediated drug delivery systems. Perhaps the most pressing of these includes the need to develop effective devices for precise targeted local delivery of ultrasound, which allow adequate doses of ultrasound to reach the targeted site. In addition, the development of intelligent sono-responsive vehicles for drug delivery is another key factor for ultrasound-mediated drug delivery to succeed. The development of smart carriers that can sequester cytotoxic anti-cancer drugs, lower their systemic side effects and hold onto the payload drug unless release is triggered by ultrasound is urgently required. Nevertheless, the use of low-power ultrasound in therapeutic medicine is in its infancy and it is expected to see a continued increase in the development of more sophisticated ultrasound-responsive drug delivery systems for individualizing chemotherapy for brain tumours and other solid human tumours.


Older adults account for a growing proportion of the surgical population. We investigated patient- and carer-reported experiences of hospitalization in this group. A mixed-methods study using telephone and postal surveys of recently hospitalized vascular surgical patients aged ≥ 65 years at an acute care academic hospital. Qualitative data were thematically analyzed using a framework approach. In total 46 patients  and nine carers were surveyed. Nine (20%) patients were frail . The majority of patients reported overall they felt their views were listened to , were kept informed  and were asked about their pain level . Among the nine carers, seven reported overall they felt their views were listened to  and were kept informed . Thematic analysis of responses to open-ended survey questions about their experience of hospitalization revealed four key themes: receiving fundamental care including hygiene and nutrition, importance of the hospital environment such as sleep and meals, being informed and involved in health care decision-making, and treating pain and deconditioning to help recovery. Findings from this study identified that older adults admitted to hospital for surgical care, and their carers, highly value care that meets fundamental needs and involves them in decision-making and recovery. These priorities can be addressed through Age-Friendly Health System initiatives such as the implementation of the “4Ms” of geriatrics care: what matters, medication, mobility and mentation.