Type 2 diabetes mellitus and cardiovascular disease and have become leading causes of morbidity and mortality among Palestinian refugees in the Middle East, many of whom live in long-term settlements and receive grain-based food aid. The objective of this study was to estimate changes in type 2 diabetes and cardiovascular disease morbidity and mortality attributable to a transition from traditional food aid to either (i) a debit card restricted to food purchases, (ii) cash, or (iii) an alternative food parcel with less grain and more fruits and vegetables, each valued at $30/person/month.n = 1,507 households) and electronic debit card delivery of food aid , and health data from a random 10% sample of refugees receiving medical care through the UN . Outcome metrics included incidence per 1,000 person-years of hypertension, type 2 diabetes, atherosclerotic cardiovascular disease events, microvascular events , and all-cause mortality. The model estimated changes in total calories, sodium and potassium intake, fatty acid intake, and overall dietary quality (Mediterranean Dietary Score [MDS]) as mediators to each outcome metric. We did not observe that a change from food parcel to electronic debit card delivery of food aid or to cash aid led to a meaningful change in consumption, biomarkers, or disease outcomes. By contrast, a shift to an alternative food parcel with less grain and more fruits and vegetables was estimated to produce a 0.08 per 1,000 person-years decrease in the incidence of hypertension , 0.18 per 1,000 person-years decrease in the incidence of type 2 diabetes (95% CI 0.14–0.22), 0.18 per 1,000 person-years decrease in the incidence of atherosclerotic cardiovascular disease events (95% CI 0.17–0.19), and 0.02 decrease per 1,000 person-years all-cause mortality (95% CI 0.01 decrease to 0.04 increase) among those receiving aid. The benefits of this shift, however, could be neutralized by a small (2%) increase in compensatory (out-of-pocket) increases in consumption of refined grains, fats and oils, or confectionaries. A larger alternative parcel requiring an increase in total food aid expenditure by 27% would be more likely to have a clinically meaningful improvement on type 2 diabetes and cardiovascular disease incidence.An individual-level microsimulation was created to estimate relationships between food aid delivery method, food consumption, type 2 diabetes, and cardiovascular disease morbidity and mortality using demographic data from the United Nations  on 5,340,443 registered Palestinian refugees in Syria, Jordan, Lebanon, Gaza, and the West Bank, food consumption data (2011–2017) from households receiving traditional food parcel delivery of food aid  among 5.3 million registered Palestinian refugees in Syria, Jordan, Lebanon, Gaza, and the West Bank.The simulation took into account the proportion who received food aid and the dietary patterns among them, including the portion of food acquired and consumed from other sources besides food aid.Using the simulation, we did not observe that a change from food parcel to electronic debit card delivery of food aid or to cash aid led to a meaningful change in consumption, biomarkers, or disease outcomes. We did observe that a shift an alternative food parcel with less grain and more fruits and vegetables may avert hypertension and type 2 diabetes but be neutralized through minimal compensatory (out-of-pocket) increases in consumption of refined grains, fats and oils, or confectionaries.A larger alternative parcel requiring an increase in total food aid expenditure by 27% would be more likely to have a clinically meaningful improvement on type 2 diabetes and cardiovascular disease.An alternative food parcel would be more effective than transitioning from traditional food aid to either debit card or cash delivery from the perspective of reducing chronic disease.But compensatory increases in consumption of less healthy foods may neutralize the improvements from an alternative food parcel unless total aid funding were increased substantially. Increasingly, refugee camps worldwide have become semipermanent or permanent, accompanied by an epidemiologic transition with fewer traumatic injuries, infectious diseases, or malnutrition as well as a much higher rate of chronic disease ,2. Type Chronic diseases affecting long-term refugee settlements are commonly nutrition-related conditions such as diabetes and cardiovascular disease; in addition, >40% of refugees  receive food aid. This aid is primarily composed of grain, flour, and rice, under a traditional model that emphasizes maintenance of calorie intake rather than dietary diversity . The reaHow much reduction in chronic disease, if any, would be expected from the proposed food aid policy changes remains unclear because field-based trials or pilot demonstration projects cannot be sustained for sufficient periods to track long-term outcomes. Increased dietary diversity, food security, sense of agency, and local economic growth have been observed from field-based randomized trials and pilot demonstration projects of alternative food aid. Diversion towards non-food products, theft, corruption, or food price inflation have been minimal or undetected in these studies, even under the cash delivery policy –16,21. HThe principal aim of this study was to identify the potential changes in type 2 diabetes and cardiovascular disease among Palestinian refugees transitioning from traditional food aid to an alternative aid . Our primary a priori hypothesis was that transitioning from traditional food parcel delivery to a debit card for food would reduce the overall incidence and complications from type 2 diabetes and cardiovascular disease, given prior evidence that the debit card intervention was particularly effective at increasing measures of dietary diversity ,16. The Approval for this study was obtained from the Institutional Review Board of Stanford University (eProtocol number IRB-39274).Study design and reporting was based on the Modeling Good Research Practice Guidelines by the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) . S1 TextAn individual-level microsimulation was created to estimate the impact of food aid delivery methods on food consumption, type 2 diabetes, and cardiovascular disease outcomes among the 20- to 79-year-old subpopulation receiving food aid (43%) among 5.3 million registered Palestinian refugees in Syria, Jordan, Lebanon, Gaza, and the West Bank. The simulation took into account the proportion who received food aid and the dietary patterns among them, including the portion of food acquired and consumed from other sources besides food aid. Individual people were simulated by sampling from the large datasets specified below to capture the correlations among demographic, food consumption, and cardiometabolic variables, including age, sex, nutrition profile, diabetes and cardiovascular disease biomarkers, chronic disease history, and medication use.The microsimulation  had the Demographic data were obtained from the UN (2017) on 5,340,443 registered refugees in Syria, Jordan, Lebanon, Gaza, and the West Bank, including age, sex, and location  and for persons obtaining electronic debit card delivery of food aid , as detailed further below.Food consumption data were obtained from the World Food Program (2011–2017) on registered refugees receiving food aid by the demographic variables listed above, categorized by quantity and caloric value of major food group procured and consumed per person per day, including aid and nonaid food  15,16].,16.15,16n = 516,386; Health data were obtained from a random 10% sample of people in the electronic health record system of the UN Relief and Works Agency , stratified by age, sex, principal diagnosis, and location  added via automated bank transfer to an electronic debit card, which could be used with an individual PIN code at participating store locations in the community. Delivery was accompanied by a text message reminder to the assigned card bearer’s phone for each household. Debit cards were electronically restricted to permit food purchases, excluding chocolates, alcohol, cigarettes, soda, or non-food items . Both exn = 2,554 households)  ). The MDChange in total calories was used to estimate changes in body mass index (BMI), using the National Institutes of Health/Hall model of body weight change, which conditions body weight change estimates on age, sex, height, and starting weight, based on results of randomized controlled trials  38–40].–40.38–40The health implications of the change from food parcel to electronic debit card delivery of food aid were estimated among a simulated refugee population of 5.3 million people. To produce the simulated population, Monte Carlo sampling with copulas was used , which iFollowing current modeling guidelines , the fuln = 1,041 households receiving a debit card compared to 1,507 receiving cash aid) and included analysis of diversion of cash for non-food uses  15.6–43.2 years), with 60.1% female and 25.3% located in Gaza, 15.1% in the West Bank, 10.2% in Syria, 8.7% in Lebanon, and 40.7% in Jordan; 43.0% were enrolled in food aid programs .Food consumption data from the World Food Program among the population of refugees receiving traditional food parcel delivery of food aid averaged 2,296 kcal/person/day , including 1,239 kcal/person/day from cereals; 137 kcal/person/day from tubers, pulses, legumes, and nuts; 230 kcal/person/day from fruits and vegetables; 343 kcal/person/day from animal products; 163 kcal/person/day from additional/added oils and fats; and 184 kcal/person/day from sugars. Food consumption among the population of refugees receiving electronic debit card delivery of food aid averaged 2,441 kcal/person/day , including 1,209 kcal/person/day from cereals; 100 kcal/person/day from tubers, pulses, legumes, and nuts; 271 kcal/person/day from fruits and vegetables; 526 kcal/person/day from animal products; 157 kcal/person/day from additional oils and fats; and 178 kcal/person/day from sugars .2 (IQR 23.5–34.1), a mean systolic blood pressure of 124.6 mmHg (IQR 111.7–136.5), a mean diastolic blood pressure of 76.5 mmHg (IQR 66.7–85.3), a mean total cholesterol of 4.9 mmol/L (IQR 4.6–5.2), a mean HDL cholesterol of 1.1 mg/dL (IQR 0.8–1.4), and a current prevalence of 12.2% for type 2 diabetes mellitus, with people with type 2 diabetes mellitus averaging a hemoglobin A1c of 8.2% (IQR 6.7–9.6)  . We noteWe did not find that the change from food parcel to electronic debit card delivery of food aid led to a meaningful change in consumption, biomarkers, or disease outcomes in our simulation model . Among t2 increase in BMI , 0.58 mmHg increase in systolic blood pressure , 0.29 mmHg increase in diastolic blood pressure , 0.06 mmol/L increase in total cholesterol , and a 0.11 mmol/L increase in HDL cholesterol , 0.77 per 1,000 person-years increase in the incidence of type 2 diabetes , 0.40 per 1,000 person-years increase in the incidence of atherosclerotic cardiovascular disease events , 0.11 per 1,000 person-years incidence in the incidence of end-stage renal disease , 0.24 per 1,000 person-years incidence in the incidence of diabetic neuropathy , 0.36 per 1,000 person-years incidence in the incidence of proliferative diabetic retinopathy , and 0.16 increase per 1,000 person-years all-cause mortality  among those receiving food aid .Among those receiving food aid in our model, a change from food parcel to cash aid was estimated to produce an overall 238 kcal/person/day increase in calorie consumption , 865 mg/person/day increase in sodium consumption , 517 mg/person/day increase in potassium consumption , 2,773 mg/person/day increase in saturated fatty acid consumption , 3,779 mg/person/day increase in monounsaturated fatty acid consumption , 1,588 mg/person/day increase in polyunsaturated fatty acid consumption , and a 0.1 point increase in the MDS on a scale from 0 to 14 such that higher values indicate better adherence to the Mediterranean diet .2 increase in BMI , 0.35 mmHg increase in systolic blood pressure , 0.23 mmHg increase in diastolic blood pressure , 0.07 mmol/L increase in total cholesterol , and a 0.17 mmol/L increase in HDL protein cholesterol .The changes in food consumption among those receiving food aid, attributable to the change from food parcel to cash aid, were estimated by our model to produce a 4.2 kg/mThe changes in biomarkers, attributable to the change from food parcel to cash aid, were estimated by our model to produce a 0.04 per 1,000 person-years increase in the incidence of hypertension , 1.28 per 1,000 person-years increase in the incidence of type 2 diabetes , 0.52 per 1,000 person-years increase in the incidence of atherosclerotic cardiovascular disease events , 0.22 per 1,000 person-years increase in the incidence of end-stage renal disease , 0.43 per 1,000 person-years increase in the incidence of diabetic neuropathy , 0.60 per 1,000 person-years increase in the incidence of proliferative diabetic retinopathy , and 0.26 increase per 1,000 person-years all-cause mortality  among those receiving aid .We observed that a food aid parcel would need to divert 8.5% of the current grain parcel to fruits and vegetables to reduce all chronic disease outcome measures while maintaining the same total aid budget, after accounting for uncertainty in fruit and vegetable procurement and delivery costs. In particular, such a shift would be expected to reduce supply of grain in food aid parcels by 40 kcal/person/day and increase fruit and vegetable supply by 7 kcal/person/day in food aid parcels. If the supply were fully consumed and did not result in any shift in non–aid-based food consumption, the change from the traditional to alternative food parcel would be expected to produce an overall 33 kcal/person/day decrease in calorie consumption , 64 mg/person/day decrease in sodium consumption , 1.3 mg/person/day decrease in potassium consumption , 345 mg/person/day increase in saturated fatty acid consumption , 358 mg/person/day increase in monounsaturated fatty acid consumption , 342 mg/person/day increase in polyunsaturated fatty acid consumption , and a 0.2 point increase in the MDS on a scale from 0 to 14 such that higher values indicate better adherence to the Mediterranean diet .The changes in food consumption among those receiving food aid, attributable to the change from traditional food parcel to alternative parcel, were estimated by our model to produce a 0.57 kg/m2 decrease in BMI , 0.12 mmHg decrease in systolic blood pressure , 0.05 mmHg decrease in diastolic blood pressure (95% CI 0.03–0.07 from a baseline of 76.5), 0.004 mmol/L decrease in total cholesterol , and a 0.03 mmol/L decrease in HDL cholesterol .The changes in biomarkers, attributable to the change from traditional food parcel to alternative parcel, were estimated by our model to produce a 0.08 per 1,000 person-years decrease in the incidence of hypertension , 0.18 per 1,000 person-years decrease in the incidence of type 2 diabetes (95% CI 0.14–0.22 from a baseline of 12.24), 0.18 per 1,000 person-years decrease in the incidence of atherosclerotic cardiovascular disease events , 0.14 per 1,000 person-years decrease in the incidence of end-stage renal disease , 0.05 per 1,000 person-years decrease in the incidence of diabetic neuropathy , 0.08 per 1,000 person-years decrease in the incidence of proliferative diabetic retinopathy , and 0.02 decrease per 1,000 person-years all-cause mortality  among those receiving aid .When we adjusted for potential mis-reporting biases in food frequency questionnaires, the results of our comparative analyses were not qualitatively different from those of the primary analysis .In the alternative food parcel simulation, we estimated that only a small degree of compensatory increase in nonaid food consumption—particularly of refined grains, oils and fats, and confectionaries—would be necessary from out-of-pocket food purchases to fully neutralize any benefits of the alternative food parcel. If refugees receiving the alternative food parcel equally increased their consumption of all 3 categories of refined grains, oils and fats, and confectionaries by only 2.1%, the cardiometabolic benefits of the alternative food parcel shown in By contrast, we estimated that an increase in the alternative food parcel budget total aid budget (including procurement and overhead costs) by 27%  would be necessary to meet the goal of an average 1.0-point absolute increase in the MDS. This increase would be accompanied by a 1.73 per 1,000 person-years decrease in the incidence of hypertension , 4.49 per 1,000 person-years decrease in the incidence of type 2 diabetes (95% CI 1.34–5.83 from a baseline of 12.24), 1.25 per 1,000 person-years decrease in the incidence of atherosclerotic cardiovascular disease events , 0.62 per 1,000 person-years decrease in the incidence of end-stage renal disease , 0.55 per 1,000 person-years decrease in the incidence of diabetic neuropathy , 1.74 per 1,000 person-years decrease in the incidence of proliferative diabetic retinopathy , and 0.55 decrease per 1,000 person-years all-cause mortality  among those receiving aid.Because transitioning from food parcel to either electronic debit card delivery or cash aid delivery of food aid was not effective at improving the chronic disease outcomes, we restricted the cost-effectiveness analysis to examining the transition from food parcels to alternative food parcels. The changes in health outcomes—attributable to a 10-year policy change from food parcels to alternative food parcels—were estimated by our model to avert 3,034 discounted DALYs per 100,000 population, over the life courses of the simulated individuals both receiving and not receiving food aid  among Palestinian refugees. One contribution of simulation models is to quantify the degree of certainty or uncertainty in making a policy decision based on available evidence. We incorporated the most comprehensive data available from the comprehensive healthcare provider for Palestinian refugees and from the main food aid providers among Palestine refugees in the Middle East; by repeatedly sampling from these data sources, we rejected our primary a priori hypothesis that transitioning from traditional food parcel delivery to a debit card for food would reduce the overall incidence and complications from type 2 diabetes and cardiovascular disease. Contrary to our hypothesis and to the supposition in the literature –24, we oWe did find, however, that transitioning to an alternative food parcel could reduce chronic disease morbidity and mortality with less uncertainty in the projected outcomes if the alternative food parcel transitioned to slightly less grain and more fruits and vegetables. We found, in particular, that small changes at the individual level in cardiometabolic risk factors would be expected to translate into larger population-level health gains and cost savings given the high prevalence of these diseases. The magnitude of the gains are somewhat higher than that anticipated, for example, from general sodium reduction in the food supply . The altOur analysis has important caveats and limitations. There is much uncertainty around our estimates due to the task of modeling long-term outcomes from shorter-term trials while accounting for the wide variation in nutrient profiles of available foods and their cardiometabolic consequences. While we focused on the chronic disease implications of transitions in food aid, there are other important reasons why food aid transitions might be important—including improved agency and choice among refugees, stimulus to local economies in semipermanent or permanent settlements, and avoidance of international dumping of undesired or extra crops under the guise of food aid . ImportaThe next logical step following this analysis is to perform field-based randomized studies of the alternative food parcel to identify unanticipated barriers to achievement, the role of seasonality and spoilage, and the degree to which refugees do or do not alter their diets in the context of an alternative food parcel. Prior to such randomized studies, our findings do not robustly support the theory that transitioning from traditional food aid to either debit card or cash delivery alone would necessarily reduce chronic disease outcomes. We did find, however, that transitioning to an alternative food parcel could be effective and should be explored further.S1 Text(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


ARID1A) or 1B (ARID1B); switch/sucrose nonfermenting (SWI/SNF)–related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 4 (SMARCA4), subfamily B member 1 (SMARCB1), or subfamily E member 1 (SMARCE1); or sex-determining region–related HMG-box 11.3Coffin-Siris syndrome  is a rare autosomal dominant syndrome characterized by impaired cognition, developmental delay, hypoplasia of the fifth digit toe nail, thick eyebrows, broad nasal bridge, wide mouth, hirsutism, microcephaly, short stature, and organ dysfunction.A 15-year-old boy presented with multiple asymptomatic cutaneous white patches, noticed by his mother 4 years before presentation. The mother's pregnancy and patient's birth history were unremarkable. The patient had difficulty feeding since birth, laryngomalacia requiring gastric tube placement, and recurrent hospitalizations for aspiration pneumonia.ARID1B gene, a premature stop codon mutation leading to a truncated protein. This mutation was not found in either parent or his 2 unaffected older sisters.His clinical features of bushy arched eyebrows, thick everted lips, broad nasal bridge, facial nevi, and developmental delay prompted metabolic and genetic testing. At 11 years old, a diagnosis of CSS was established via whole-exome sequencing, revealing a de novo mutation  in the Clinical examination showed distinctive coarse facial features , fifth fARID1A, ARID1B, SMARCA4, SMARCB1, SMARCE1) encode for the Brahma-associated factor complex.p53 mutations.ARID1 pathogenic variant),ARID1B),SMARCA4), meningiomas, and schwannomatosis (SMARCB1 pathogenic variant).5Most of the 6 identified genes causative of CSS  gene,MITF that would critically cause its dysfunction. Herein, we suggest that the mutations in ARID1A, ARID1B, SMARCA4, SMARCB1, and SMARCE1 (causative of CSS) might create some interference that would lead to disturbances in the interaction between the SWI/SNF activity and MITF and alterations in MITF-regulated processes, such as the transcription of the 3 main enzymes involved in melanogenesis  gene We report a patient with a genetically confirmed diagnosis of CSS and a striking phenotype affecting melanocytes, namely vitiligo and numerous melanocytic nevi. CSS requires a multidisciplinary and supportive approach. There are limited treatment options for vitiligo, with the objective to slow disease progression and promote repigmentation. For this patient, sunscreen and topical glucocorticoids were recommended. His laboratory studies were negative for thyroid disease, pernicious anemia, Addison disease, and multiple endocrinopathy syndrome. His family opted to pursue molecular analysis of his nevi to find if other predictors of malignancy are present. Those studies are pending as of the publication date of this report.ARID1B mutation, vitiligo, and melanocytic nevi might facilitate identification of therapeutic targets for disorders with aberrant function or proliferation of melanocytes.Our findings are relevant for pediatric patients presenting with vitiligo and suggest a higher index of suspicion for the risk of melanoma when melanocytic nevi are present. Last, the link between


Significance: NADPH oxidases (Noxs), of which there are seven isoforms , are professional oxidases functioning as reactive oxygen species (ROS)-generating enzymes. ROS are signaling molecules important in physiological processes. Increased ROS production and altered redox signaling in the vascular system have been implicated in the pathophysiology of cardiovascular diseases, including hypertension, and have been attributed, in part, to increased Nox activity.Recent Advances: Nox1, Nox2, Nox4, and Nox5 are expressed and functionally active in human vascular cells. While Nox1, Nox2, and Nox4 have been well characterized in models of cardiovascular disease, little is known about Nox5. This may relate to the lack of experimental models because rodents lack NOX5. However, recent studies have advanced the field by (i) elucidating mechanisms of Nox5 regulation, (ii) identifying Nox5 variants, (iii) characterizing Nox5 expression, and (iv) discovering the Nox5 crystal structure. Moreover, studies in human Nox5-expressing mice have highlighted a putative role for Nox5 in cardiovascular disease.Critical Issues: Although growing evidence indicates a role for Nox-derived ROS in cardiovascular (patho)physiology, the exact function of each isoform remains unclear. This is especially true for Nox5.Future Directions: Future directions should focus on clinically relevant studies to discover the functional significance of Noxs, and Nox5 in particular, in human health and disease. Two important recent studies will impact future directions. First, Nox5 is the first Nox to be crystallized. Second, a genome-wide association study identified Nox5 as a novel blood pressure-associated gene. These discoveries, together with advancements in Nox5 biology and biochemistry, will facilitate discovery of drugs that selectively target Noxs to interfere in uncontrolled ROS generation. Reactive oxygen species (ROS) are signaling molecules that influence gene expression, oxygen sensing, cell growth, and cell death. They define the cellular phenotype by regulating functions such as migration, contraction, differentiation, and secretion . ROS hav2 to produce superoxide (NADPH +2O2 → NADP+ + H+ + 2O2−), which can dismute to generate secondary species, including hydrogen peroxide (H2O2) and hypochlorous acid, which together play a role in host defense by killing or damaging microbes -induced hypertension in rodents, production of O reduced , 95, 96. reduced . Rats wisynthase , 133. Nosynthase  and seemsynthase .While extensive experimental data support a causative role for oxidative stress in hypertension, there is a paucity of evidence for a direct role of ROS in the pathophysiology of human hypertension , 84, 1012O2 generation  is essential for Nox5 activation, as evidenced in cell studies where ROS are not generated in Ca2+-free buffer , is the most recently discovered Nox and is unique  20). No. No20). e buffer . Becausetallized .Unlike p22phox-regulated vascular Noxs , Nox5 is expressed primarily in intracellular compartments localized mainly in the perinuclear area and endoplasmic reticulum (ER)  66). In. In66). 2+, thereby facilitating ROS generation at lower Ca2+ levels , extracellular signal-regulated kinase 1/2 (ERK 1/2), and tyrosine, influence phosphorylation of Nox5 . Nox5 phosphorylation increases sensitivity to Ca+ levels . Numerou of Nox5  , and palmitoylation . HoweverThe Nox5 gene is expressed in a variety of fetal tissues and in adult testes, spleen, lymph nodes, pancreas, placenta, bone marrow, uterus, kidney, stomach, and cancer cells. It is also present in cells of the cardiovascular system, including cardiomyocytes, and endothelial and vascular smooth muscle cells , 73. In 2+]i in Nox5 regulation, and the critical role of Ca2+ in controlling the contractile machinery in muscle, we questioned whether Nox5 plays a role in vascular contraction. Our recent data show that in human vascular smooth muscle cells, Nox5 regulates procontractile signaling pathways, effects that are attenuated in siRNA Nox5-silenced human vascular smooth muscle cells ., 81.2+]iThe possible role of Nox5 in contraction was previously highlighted in arthropod models, which express an ortholog of Nox5, called dNox , 99. Depet al. . Th. Th61). One of the earliest studies suggesting a link between vascular NOX5 and human cardiovascular disease was performed in coronary arteries in patients undergoing heart transplantation . In pati2+ may be especially important in the heart and coronary circulation where cardiovascular contractility is highly regulated. Studies in porcine coronary arteries demonstrated that Nox5 plays an important role in regulating the potassium intermediate/small-conductance calcium-activated channel, subfamily N member 4 (KCNN4), which seems to be important for coronary artery smooth muscle cell phenotypic modulation, contraction, and progression of atherosclerosis physiology, Nox5 has been implicated to play an important role in various physiological and pathological processes .2O2 increased sperm motility, effects that were dependent on Nox5-induced ROS generation  and may relate, in part, to the lack of rodent experimental models that express Nox5. However, studies in transgenic mice that express human Nox5 and investigations in larger mammals that contain Nox5 endogenously, including rabbits and primates, will shed light on the physiological and pathological significance of Nox5. Moreover, studies in humans in health and disease will unravel the clinical role of Nox5. The recent GWAS highlighting the association between the Nox5 gene and hypertension is certainly interesting and provides a credible foundation for further investigation of Nox5 in human cardiovascular disease.The importance of Noxs in ROS generation in the cardiovascular system and other systems is now clear. Nox isoforms are differentially regulated. Whereas Nox1, Nox2, and Nox4 have an obligatory need for p22phox for their activation to generate ROS, Nox5 is p22phox independent and produces O


Objective: To evaluate the efficacy and treatment rationale of Hürthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct genetic make-up and the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kiase (PI3K)/AKT signaling pathways are potential targets for anti-cancer agents in the management of recurrent HCC. The presence or absence of gene variants can give a rationale for targeted therapies that could be made available in the context of drug repurposing trials. Methods: Treatment included everolimus, sorafenib, nintedanib, lenvatinib, and panitumumab. Whole genome sequencing (WGS) of metastatic tumor material obtained before administration of the last drug, was performed. We subsequently evaluated the rationale and efficacy of panitumumab in thyroid cancer and control cell lines after epidermal growth factor (EGF) stimulation and treatment with panitumumab using immunofluorescent Western blot analysis. EGF receptor (EGFR) quantification was performed using flow cytometry. Results: WGS revealed a near-homozygous genome (NHG) and a somatic homozygous TSC1 variant, that was absent in the primary tumor. In the absence of RAS variants, panitumumab showed no real-life efficacy. This might be explained by high constitutive AKT signaling in the two thyroid cancer cell lines with NHG, with panitumumab only being a potent inhibitor of pEGFR in all cancer cell lines tested. Conclusions: In progressive HCC, several treatment options outside or inside clinical trials are available. WGS of metastatic tumors might direct the timing of therapy. Unlike other cancers, the absence of RAS variants seems to provide insufficient justification of single-agent panitumumab administration in HCC cases harboring a near-homozygous genome. BRAF mutated PTC and HCC [Differentiated non-medullary thyroid cancers (DTC), account for the vast majority ~95%) of thyroid cancers. These can be divided into main histologic types papillary (PTC), follicular thyroid carcinoma (FTC) including subtype variants, and Hürthle cell carcinoma (HCC) ,2. Usual% of thyr and HCC ,9,10. HC and HCC , but ran and HCC ,12,13,14Typically, HCC features include highly eosinophilic cytoplasm due to accumulated dysfunctional mitochondria ,16 with RAS [RAS mutations dually activate the MAPK and PI3K-AKT pathways. In thyroid carcinogenesis, the latter appears to be more preferred as suggested by the association with AKT activation [Major drivers of thyroid cancer include the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kiase (PI3K)/AKT signaling pathway ,25,26,27RAS . RAS muttivation ,30. Strotivation . In a retivation . At the tivation ,21,27,33tivation ,33.In the course of disease, systemic or molecular-targeted therapies may be considered . For exaKRAS or NRAS genes and target the extracellular part of EGFR [RAS wildtype non-CRCs is part of the DRUP trial. In this study, we examined the clinical effects of several targeted drugs in a patient with HCC for which whole genome sequencing of metastatic tumor material was performed at an advanced stage of his disease. With HCC reportedly having lower prevalence rates of RAS mutation than FTC [RAS and was considered for panitumumab therapy. Considering the background of HCC, the impact of panitumumab on its receptor of target and downstream major signaling activators, AKT and ERK, involved in thyroid tumorigenesis, was analyzed in an HCC cell line model with a NHG.In the Netherlands, the Drug Rediscovery Protocol (DRUP) basket trial (NCT02925234) was initiated for selecting patients to be administered off-label targeted therapies based on their tumoral mutational profiling, rather than the regular indication range concerning the cancer type it is originally registered for. As such, panitumumab and cetuximab are part of the standard care for advanced colorectal cancer (CRC) ,38. Thes of EGFR . For thathan FTC ,32,40,41Possible treatment options for recurrent HCC are illustrated by a 40-year-old male patient with a left sided widely invasive HCC (5.3 cm in diameter) with extensive vaso-invasion. No lymph node metastasis was found (0/17). The patient had no known risk factors, no intoxications, and his family history included no cancers except for one third-degree family member who was curatively treated for thyroid cancer at the age of 39. Surgical resection of the thyroid with lymph node dissection, radio-iodide therapy, and TSH suppression was performed five years prior to his death. On pre-operative and post-operative I-131 therapy scan, iodide accumulation was detected in the bone, which is indicative of metastases. Five months after this radio-iodide therapy, Positron emission tomography-computed tomography (PET-CT) showed vitality of these bone metastases with rising thyroglobulin, which comply with radio-iodide refractory disease. For the metastatic osseous lesion, the patient received radio-frequent ablation and cementation, which was followed by radiotherapy. Six months later, a PET-CT scan showed residual/recurrent metastasis at the same osseous site with additional novel metastatic lesions in the vertebral column (C7) and liver.The patient was consecutively treated with various tyrosine kinase inhibitors  in or out of clinical trials with consideration of clinical status along with assessment of overall lesion response . At thisUpon giving everolimus, there was a stable disease at 12 weeks. Adverse effects included mild fatigue, rash, acne, moderate mucositis and were manageable by using local triamcinolone, lidocaine, and daktarin gels. However, progressive disease was observed at 25 weeks, which initiated a switch to sorafenib. After one week, the patient developed signs of intolerable toxicity with severe palmo-plantar dysesthesia syndrome with severe blistering, legitimizing discontinuation followed by restart at a reduced dose, which reiterates toxicity and ditto implications. On nintedanib, a seven-week course of stable disease was followed by progressive disease at 16 weeks. Adverse events included mild fatigue and alopecia. The patient also received radiation on a vertebral metastatic lesion and stereotactic radiotherapy on a new extra-axial dural lesion that developed before the initiation of nintedanib. On lenvatinib, a partial response was seen over one year of treatment, which was his best overall response (BOR). From lenvatinib use, hypertension ensued initially without need for antihypertensive drugs. Other adverse events included mild diarrhea, fatigue, and raised TSH levels (acted upon by adjustments of the levothyroxine dose). Five months further, progressive disease was determined. However, 10 months of continued use was added out of clinical benefit. Bone metastases had become more expansive and liver metastases were progressive as well, whereupon lenvatinib was ceased eventually. On the vertebral lesion (C7), embolization was done, when the risk of inflicting harm to the myeloid would be too high for radiotherapy.In the context of the DRUP trial, whole genome sequencing (WGS) was performed on biopsy material from a liver metastasis, which showed loss of heterozygosity (LOH) of whole chromosomes on all chromosomes except for chromosome 5, 7, and 20 .TSC1  and in NF1 . Other detected homozygous somatic variants included TP53  and RB1  During the molecular tumor board meeting both the TSC1 and NF1 variants were considered a potential candidate target rendering sensitivity to mTOR inhibition . As opposed to the metastatic lesion, next-generation sequencing (NGS) of the primary tumor did not reveal the TSC1 variant. In addition, in contrast to the metastatic lesion after a five-year disease interval, the primary tumor appeared heterozygous for chromosome 12 based on a SNP in CDKN1B ), whereas the metastatic hepatic lesion revealed homozygosity.We subsequently sequenced the primary tumor, which harbored the same homozygous RAS variants motivated the prescription of panitumumab, which was made available within the DRUP basket trial. Two cycles of panitumumab were administered, but no clinical improvement was noted. The patient was quickly progressive during the panitumumab regimen and his clinical state deteriorated on a pathologic vertebral fracture.The absence of TP53 variant ), the mutational load was low (11.02 Mutations/Mb) and no other pathogenic variants of gene fusions were found. These were compared to thyroid cancer cell lines SW579, BHP 2-7, and TT2609-C02.We further addressed the occurrence of non-responsiveness in the HCC index patient toward the EGFR receptor directed inhibition therapy using panitumumab in thyroid cancer cell line models for HCC or FTC with an NHG. These are cell lines XTC.UC1, FTC-236 [EGFR was quantified using flow cytometry using A431 and C33A cells as a positive and negative control, respectively. The TC cell lines all expressed the EGF-receptor on their cell membranes and could be subdivided into two groups: one group (SW579 and XTC.UC1) with values ranging from approximately 120,000 to 135,000 MEF and a second group  with values ranging from approximately 30,000 to 50,000 MEF. A plateau phase was reached at approximately 2 μg panitumumab .NRAS mutation. Panitumumab significantly reduced the levels of EGFR phosphorylation to control levels. Panitumumab alone did not alter the phosphorylation status of the EGF receptor (control). EGF induced ERK phosphorylation in FTC-236, XTC.UC1, SW579, and in the control cell line A431. However, BHP 2–7 (with a RET/PTC1 rearrangement) and TT2609-C02 (harboring a NRAS mutation) seem to remain unaffected by EGF or panitumumab regarding ERK phosphorylation. These cell lines show constitutive ERK phosphorylation irrespective of the treatment. Panitumumab could only partly inhibit EGF-induced ERK phosphorylation. Additionally, AKT is constitutively active in these cell lines, at low levels in TT2609-C02. EGF induced additional AKT phosphorylation in XTC.UC1 and in A431. Of interest, AKT phosphorylation was significantly higher in TC cell lines FTC-236 and XTC.UC1, that harbor a near-homozygous genome (NHG) [Comparable to flow cytometry, Western blotting showed EGFR overexpression in A431 cells (positive control) as compared to all thyroid cell lines, with XTC.UC1 and SW579 showing relatively more EGFR compared to BHP 2-7, FTC-236, and TT2609-C02 . Prior tIn the present study, we illustrate the treatment options for patients with recurrent and metastatic HCC of the thyroid (with NHG) following the targeted treatments chosen for a single HCC patient with metastatic disease.Various targeted agents  were administered, based on approved status, or availability within the frame of trials, consecutively, in a time frame of four years. Because of a quick clinical progression due to metastasis, the patient’s death was within five years after surgery.Everolimus has been studied in thyroid cancer patients in phase II trials showing a median PFS of 11.8 months   at 14.25 months as the best overall response. This multi-kinase inhibitor is one of the approved drugs for the treatment of progressive, refractory differentiated thyroid cancer. In the randomized phase III SELECT trial, lenvatinib has been shown to increase progression-free survival (PFS) with 14.7 months compared to a placebo, a 64.8% response rate, and an 87.7% disease control rate .When exploring potential further directions for personalized therapy based on molecular profiling in the current patient, whole genome sequencing was performed on metastatic biopsy material. HCC carrying a near-homozygous genome was revealed, with loss of heterozygosity on all autosomes except for chromosomes 5, 7, and 20. Tumors with extensive NHG, which is a characteristic HCC feature ,19,20,21TSC1 mutation, was detected upon WGS analysis of metastatic material. Various cancer types have been shown to carry druggable targets for mTOR inhibitors, including mutations in MTOR, TSC1, TSC2, NF1, PI3KCA, PIK3CG, STK11, and RHEB [TSC1 nonsense mutation, showing clinical benefit to everolimus [TSC2 nonsense mutation rendering striking sensitivity to everolimus with an 18-month response. Progressive disease followed due to an acquired mutation in MTOR, which confers resistance to allosteric mTOR inhibition [Furthermore, a potential target for mTOR inhibitory treatment, the homozygous and RHEB ,52,53,54TSC1 homozygous pathogenic variant (on chromosome 9) detected on metastatic material, was not present in the primary tumor. This would serve as a possible explanation for the patient’s relative unresponsiveness to everolimus earlier in the disease course. In retrospect, the revelation of the newly acquired homozygous TSC1 variant in the hepatic metastasis could have justified a second attempt for an everolimus regimen. However, the patient’s condition did deteriorate too rapidly for such a choice and everolimus was not available within the DRUP protocol.Furthermore, in the metastatic material from the present patient, chromosome 12 revealed LOH, as opposed to the primary resected lesion five years prior. Presumably, this is indicative of the gradual progression of whole chromosome losses, as seen in this type of tumors. With chromosomes 5, 12, 20, and especially chromosome 7 often being exempt from LOH ,19,20,21RAS status prompted the initiation of panitumumab. A fully humanized IgG2 monoclonal antibody with high affinity and specificity for EGFR, which is part of standard treatment for metastatic CRC [Confirmation of wild-type atic CRC ,38. HoweThe outcome of the cell line experiments may serve as a rationale against anti-EGFR therapy with panitumumab in HCC patients. The non-effectiveness of anti-EGFR receptor treatment might be caused by constitutive AKT signaling especially in XTC.UC1 and FTC-236, that serve as a model for HCC with NHG. Panitumumab, however, appeared to lower ligand stimulated expression of activated ERK, but not to basal levels, which is suggestive of the presence of orphan receptor mechanisms .RAS mutation, these cases could be considered unlikely to achieve a potential benefit from panitumumab treatment. The study by Ganly et al. also reports AKT/mTOR activation in HCC [PTEN, PIK3CA, AKT1, NF1, and RAS [PTEN activity [PTEN is located on, may also lead to a decrease in PTEN activity.To the best of our knowledge, panitumumab has not been tested before in HCC and FTC cell line models harboring a near-homozygous genome in particular. Indeed, strong PI3K/AKT signaling and phosphorylated AKT (pAKT) overexpression has been reported in HCC and, moreover, found to differentiate widely invasive HCC from adenoma, which appears to be a potential mechanism in HCC development . Thus, en in HCC . Tumors n in HCC ) of chron in HCC ,33. Seve and RAS ,21,27,33activity , an NHG Although the case described involves only one patient, it is rather illustrative of the management course using targeted therapies, considerations, and dilemmas encountered in the application thereof. Options provided by drug repurposing for developing a tailored treatment plan based on molecular genetic analysis, is demonstrated. Simultaneously, the elucidation of insight into the molecular characteristics in cancer types may serve to be crucial in this process as well in order to define the ultimate range of indications. The patient succumbed to his disease at a relatively young age, within five years of a refractory HCC, which is also an exemplification of the poor prognosis of refractory HCC and emphasizes the pressing need for effective therapy.The patient presented was enrolled under an earlier study protocol for the administration of everolimus as part of the THYRRAD study approved by the Institutional Review Board of the Leiden University Medical Center  and registered at ClinicalTrials.gov  and for nintedanib . Written informed consent was provided by the patient. Formalin fixed paraffin embedded (FFPE) primary thyroid carcinoma samples of the primary study were available. Sorafenib was used as standard therapy and lenvatinib was provided by a Named Patient Program (NPP). Panitumumab was made available to the patient as part of the DRUP trial . Assessment of overall lesion response was done using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 .https://emea.illumina.com/company.html#) at the Hartwig Medical Foundation. For WGS analysis, a fresh-frozen biopsy was taken from a metastatic liver lesion following lenvatinib treatment, together with a control blood sample. WGS DNA sequencing was performed and analyzed following standardized procedures described by Priestley et al. [Tissue biopsy material from the liver metastasis was analyzed using whole genome sequencing (WGS)  Assay and estimation of Tumor mutational burden (TMB) (thermofisher.com/tmb). Isolated RNA from the XTC.UC1 cancer cell line was analyzed using NGS with the Archer FusionPlex CTL Panel for the detection of fusions in target genes ALK, AXL, BRAF, CCDN1, FGFR1-3, MET, NRG1, NTRK1-3, PPARG, RAF1, RET, ROS1, and THADA (https://archerdx.com/fusionplex-assays/ctl-rna).Isolated DNA from the XTC.UC1 cancer cell line was analyzed using NGS with the OncominePathogenicity of DNA variants is annotated in classes, ranging from class 1 to 5 . Class 5The following human differentiated thyroid cancer cell lines were used for assessing panitumumab treatment on EGFR and downstream signaling using Western blot analysis: BHP 2–7, SW579, XTC.UC1, FTC-236, and TT2609-C02 .As a type of control, the epidermoid carcinoma cell line with known EGFR overexpression, A431 cells, was used in this study.® 10 system, Promega, Leiden, The Netherlands).For assessing EGFR quantification using flowcytometry, an additional cell line with known lack of EGFR expression, C33A, was used as a control . Cell li2, 95% air, 37 °C). RPMI medium 1640  was used for SW579 cells and TT2609-C02 cells. The other cell lines were maintained in DMEM/F-12 medium . Culture media were supplemented with 10% heath-inactivated Fetal Bovine Serum , penicillin (50 U/mL), streptomycin (50 µg/mL) .The cell lines were cultured under standard conditions in humidified atmosphere  and harvested using HBSS containing 0.125% trypsin  and 0.5 mM EDTA at 37 °C. For DNA isolation, the NucleoSpin purification kit  was used on cell pellets, according to the manufacturer’s instructions. DNA concentrations were determined using the Picogreen method (Life Technologies).Cell counts were performed using AO-DAPI , diluted 1:20. Each cell line was loaded in quadruplicate using the NC-Slide A8  and read out by Automated Cell Analyzer NucleoCounter NC-250  with NucleoView NC-250 software .Cells were cultured until 70% to 80% confluency. Next, cells were serum starved, stimulated by EGF alone for 10 min, or treated with panitumumab  for 1 h (20 µg/mL) and stimulated with EGF  for 10 min (100 ng/mL), which is followed by washing with ice cold PBS and lysed with Hot-SDS buffer containing PhosSTOP .TM Protein Assay, according to the manufacturer’s instructions . Next, 10 µg lysates were mixed with 4× Laemmli Sample Buffer  containing βME, and heated for 5 min at 100 °C and loaded onto a 1.5 mm 10% acrylamide gel in addition to molecular weight markers . Electrophoresis was performed at 50 V throughout the gel. For blotting, the Bio-Rad semi-dry Trans-Blot Turbo Transfer System was used . Next, blots were washed in 1×TBS, blocked in Odyssey Blocking Buffer (TBS)  with 1× TBS in ratio 1:1 for 1 h, washed in 1× TBS, and incubated overnight at 4 °C with antibodies in primary antibody diluent containing 1× TBS, 0.1% Tween-20, 5% BSA . The next day, blots were washed in 1× TBS and incubated for one hour with the secondary antibodies in 1× TBS/0,1% Tween-20/5% BSA protected from light.Protein concentrations were determined with the Bio-Rad DCAfter washing with 1× TBS, the blots were air dried and imaged on high-resolution with the Odyssey Infrared Imaging System , using intensities 1.5 and 3.0 for the fluorescent color channels red (700) and green (800), respectively. Images were analyzed using Image Studio Lite Ver 5.2 software package .Primary antibodies: anti-αTubulin Mouse, 1:100,000 ; EGFR Rabbit mAb, 1:4000 . pEGFR (Tyr1148) Rabbit mAb, 1:1000 . Akt Mouse mAb, 1:2000 . pAkt (Ser473) Rabbit mAb, 1:1000 . Erk 1/2 Mouse mAb, 1:1000 . pErk 1/2 (Thr202/Tyr204) Rabbit mAb, 1:2000 . Secondary antibodies: green-fluorescent Goat anti-Rabbit IRDye 800CW, 1:10,000 . Red-fluorescent Goat anti-Mouse IRDye 680LT, 1:10,000 .Cells (0.5 million/test tube) were spun down . Fifty µL in PBS/1%BSA (1% PBA) diluted panitumumab (range 1 ng–2 µg) was added to the cell pellets and gently vortexed. Cells were incubated for 60 min on ice. Next, cells were washed twice with PBS and a secondary reagent was added  to the pellet, mixed, and incubated for another 60 min on ice in the dark. Cells were washed twice with PBS. Lastly, 500 µL of 1% PBA was added containing 1 µM of propidium iodide staining dead cells, vortexed, and stored on ice in the dark for 30 min prior to the analysis on an LSRII flow cytometer (BD Biosciences). A blue 488 nm 20 mW laser was used for excitation. A threshold was set on the FSC detector. The FSC-W vs. FCS-H and SSC-W vs. SSC-H were used to gate out doublets during acquisition. For quantitative flow cytometry, Cyto-CalTM calibration beads  were used according to the manufacturer’s instructions, which allows the presentation of EGFR expression in terms of molecular equivalent fluorochrome (MEF). FITC fluorescence or green fluorescence equivalent (beads) was collected using a 530/30 band pass filter. An FSC-A vs. PI-A fluorescence life gate was used for dead cell discrimination and 20,000 single live cell events were collected for each measurement. A data file contained all events, including debris and aggregates. Data were analyzed using WinList 8 .RAS wildtype status for administration seems insufficient for these recurrent thyroid cancer cases. This study further adds to the definition of the indication range of molecularly targeted therapies in precision oncology, which evolved from an increasing knowledge on cell signaling mechanisms involved in different types of cancer. Moreover, it highlights the use of broad genomics assessment for developing a personalized approach.In this study, we described current treatment options for recurrent HCC illustrated by a patient and, in addition, assessed the potential value of panitumumab on major signaling pathways involved in thyroid cancer in surrogate cell line models. Based on the results of this study, we conclude that recurrent HCC cases do not seem likely to benefit from panitumumab and the criterion of


In the presence of Acyl-CoA, lysophosphatidylcholine acyltransferase (LPCAT) converts LPC to PC, which rapidly gets recycled by the Lands cycle. However, overexpression or enhanced activity of PLA2 increases the LPC content in modified low-density lipoprotein (LDL) and oxidized LDL, which play significant roles in the development of atherosclerotic plaques and endothelial dysfunction. The intracellular enzyme LPCAT cannot directly remove LPC from circulation. Hydrolysis of LPC by autotaxin, an enzyme with lysophospholipase D activity, generates lysophosphatidic acid, which is highly associated with cancers. Although enzymes with lysophospholipase A1 activity could theoretically degrade LPC into harmless metabolites, they have not been found in the circulation. In conclusion, understanding enzyme kinetics and LPC metabolism may help identify novel therapeutic targets in LPC-associated diseases.Lysophosphatidylcholine (LPC) is increasingly recognized as a key marker/factor positively associated with cardiovascular and neurodegenerative diseases. However, findings from recent clinical lipidomic studies of LPC have been controversial. A key issue is the complexity of the enzymatic cascade involved in LPC metabolism. Here, we address the coordination of these enzymes and the derangement that may disrupt LPC homeostasis, leading to metabolic disorders. LPC is mainly derived from the turnover of phosphatidylcholine (PC) in the circulation by phospholipase A These two pathways are part of the Lands cycle [Lysophosphatidylcholine (LPC), also called lysolecithins, is a class of lipid biomolecule derived by the cleaving of phosphatidylcholine (PC) via the action of phospholipase A2 (PLA2) ,2 and/or2 (PLA2) . In heal2 (PLA2) ,5,6. PC,ds cycle , which ids cycle , liver [ds cycle , and adids cycle , but the2 (Lp-PLA2) in circulation [In the liver, LPCs upregulate genes involved in cholesterol biosynthesis and downregulate genes involved in hepatic fatty acid oxidation . Higher culation ,29. In cGpr17) signaling. Highly expressed in brain tissue, GPR17 reduces the intracellular cAMP level and induces pro-apoptotic gene XIAP-associated factor 1 (Xaf1) expression, which in turn inhibits oligodendrocyte survival and precursor cell differentiation [LPC activates multiple signaling pathways that are involved in oxidative stress and inflammatory responses. The signaling cascade is triggered through G protein–coupled receptor G2A ,31,32,33ntiation . In anotntiation . LPC medntiation . The mecntiation .As described above, LPC may alter the physiology of the vascular endothelium, pericytes, and neuron cells in vitro and in vivo, indicating it may be a compelling risk factor and may be associated with the pathogenesis and prognosis of cardiovascular diseases. However, findings from recent clinical lipidomics studies have been controversial and somewhat confusing. For example, plasma LPCs showed an inverse relationship with cardiovascular diseases ,52,53. I2 and LPC content [Cardiovascular diseases are a class of diseases that include atherosclerosis, diabetes, metabolic syndrome, myocardial infarction, and angina. According to the World Health Organization, cardiovascular diseases account for 17.7 million deaths  each year and are now the leading cause of death in the world . Atheros content . Interes content ,52,53. I content ,71,72,731–42 peptide oligomer formation and neurotoxic protein aggregation, indicating that inhibiting LPC generation may be important in treating neurodegenerative diseases [Oligodendrocytes are myelin-producing cells that provide metabolic support for neurons and prevent neurodegeneration . Myelinadiseases ,81. In odiseases . Howeverdiseases ,55 and tdiseases ,77.Through Notch1 and/or ERK1/2 signaling, LPC induces monocyte chemoattractant protein-1 and inflammatory cytokine expression and damages endothelial cells ,19,50. I2 , did not yield promising results in clinical trials [2 has the anti-inflammatory function of degrading PAF [2 may involve the use of combined RNA interference (RNAi), which ameliorated atherosclerosis in apolipoprotein E-deficient mice [PC synthesized in the liver is the most abundant lipid component (up to 70% mole ratio) of plasma very low density lipoprotein (VLDL) and also makes up close to 40% of nascent high-density lipoprotein (HDL) ,84. In t2 . LPC is y system . ApoCIIIpression ,92,93. Ipression . Extensir events ,95,96,97diseases ,99,100. morrhage ,102,103.l trials ,105. Altding PAF ,107 and ding PAF ,108. Anoent mice .LCAT is secreted from the liver and catalyzes the transfer of the fatty acids at position sn-2 of PC to free cholesterol in plasma, which results in the formation of cholesterol esters and LPC on the surface of HDL and LDL ,111 Fig. In the O-GlcNAc glycosylation and phosphorylation of the p38 mitogen-activated protein kinase [Swe/PSIdE9 mice, chronic intermittent hypoxia triggered earlier learning memory impairment. In a symptomatic N5 TgCRND8 mouse model of Alzheimer’s disease, cytosolic phospholipase A2α activity progressively increased; overall LPC levels progressively rose. The authors concluded that disruptions in Lands cycle metabolism were linked to the onset of symptoms and a progressive behavioral decline in mice with pre-existing Aβ pathology [Hypoxia induces a reprogramming of cell respiration and carbohydrate metabolism . Under hn kinase . Some lin kinase . In addin kinase . In AbPPathology . HoweverPlasma LPC is rapidly cleared from circulation by transporters such as albumin and alpha-1 acid glycoprotein (AGP) to the liver for the synthesis of PC , or it aAbout 80% of LPC is bound to albumin ,143. HypThe concentration of AGP in the plasma increases under inflammatory conditions. Similar to the function of antitrypsin, AGP exerts anti-inflammatory effects by inhibiting platelet aggregation, preventing superoxide production, and attenuating TNF-α effects on cells . In addiP4-ATPases are lipid flippases expressed on the eukaryotic plasma membrane. They function in translocating phospholipids from the exoplasm to the cytosolic area against a concentration gradient via ATP hydrolysis ,150. In 2+ influx and NO synthesis [Human lipoproteins, including VLDL, LDL, and HDL, contain LPC. However, the levels of LPC in these lipoproteins are debated ,156,157.ynthesis , and proynthesis . These pynthesis ,163.LPCAT1 gene may contribute to the progression and metastasis of human cancers, such as hepatocellular carcinoma [LPCAT2 gene is upregulated in breast and cervical cancers [LPCAT3 is regulated by peroxisome proliferator-activated receptor δ. Transient liver-specific knockdown of LPCAT3 in mice attenuated the fatty acid metabolic pathway [LPCAT3 knockdown resulted in LPC accumulation in the liver but promoted VLDL secretion and microsomal triglyceride transfer protein expression [LPCAT3 deficiency reduced lipid adsorption in small intestine [Under normal physiological conditions, LPC is cleared by enzymes such as acyl-CoA:LPCAT , locatedarcinoma , oral sqarcinoma , breast arcinoma , prostatarcinoma , and colarcinoma . LPCAT2 arcinoma . Express cancers . LPCAT3  pathway ,165. In pression . In addintestine . LPCAT4 ntestine . In colontestine . Tumor nntestine ,179.1, C, or D, according to the cleavage site  was first cloned from human brain tissue [LYPLA2 gene) is a cytosolic enzyme that is transported through the cell membrane by palmitoylation [1 (lysoPLA1), which is classified as EC 3.1.1.5. Instead, the depalmitoylating activity of APT-1/APT-2 is associated with membrane protein localization and signaling such as Ras [Lysophospholipase I  and removal of the choline moiety from LPC by autotaxin [1–6 or LPAR1–6, involved in autotaxin–LPA axis signaling [LPA signaling and function: The LPA concentration in plasma ranges between 100 to 164 nM, which is about 1000 times less than that of LPC ,211,212.utotaxin ,214. Plautotaxin . There aignaling ,217. LPAignaling . LPA plaignaling  and stimignaling .LPA and diseases: LPA signaling is associated with metabolic and inflammatory disorders including obesity, insulin resistance, atherosclerosis, and myocardial infarction ,222,223.2 for LPC production. LPCAT contributes to reducing LPC levels; however, the overexpression of LPCAT is associated with cancer. Direct degradation of LPC by autotaxin with lysophospholipase D activity produces LPA, which is another mediator that is highly associated with cancers. Enzymes with lysophospholipaseA1 activity can degrade LPC into harmless materials, but no enzyme with strong lysophospholipase A1 activity has been identified. Targeting LPC may be an important therapeutic option for treating cardiovascular and neurodegenerative diseases. The use of RNAi to inhibit Lp-PLA2 or enzyme lysophospholipase A1 activity may potentially be an effective future strategy. In summary, gaining a better understanding of the enzymes and non-enzyme proteins involved in LPC metabolism and how their levels correlate with disease conditions may be useful in identifying novel therapeutic targets for LPC-associated diseases.LPC, via G protein-coupled receptor signaling, has harmful effects on various cells that include enhancing inflammatory responses, disrupting mitochondrial integrity, and inducing apoptosis. However, the optimal level of LPC in the plasma has not been established, and the mechanisms underlying LPC’s harmful effects are not well understood. Levels of LPC in LDL positively correlate with disease development. An increase in LPC levels is determined primarily by enzyme activity—Lp-PLA


Postoperative cognitive dysfunction (POCD) is serious disorder in the central nervous system common in aged patients after anesthesia. Although its clinical symptoms are well recognized, however, the molecular etiology of the POCD remains unrevealed. Similarly, neither gold standard molecular diagnosis nor effective treatment is available for POCD until the present. Therefore, we aimed to explore the molecular mechanism of this disorder through investigating lncRNAs and mRNAs associated with POCD human patients and investigate their underlying regulatory pathways. In this study, we recruited 200 patients requiring hip or knee replacement surgery. Their neurological functions were assessed at two time points, 1 day before the surgery and 30 days post-surgery. In parallel, serum samples were collected from the participants to analyze lncRNAs and mRNAs differential expression profile between POCD and non-POCD patients using microarray analysis. To further investigate the role differentially expressed mRNA and lncRNAs, Gene Ontology (GO), pathway analyses on mRNAs and lncRNA-mRNA interaction network were performed. As a result, 68 lncRNAs and 115 mRNAs were dysregulated in the POCD group compared to non-POCD group. Among them, the top 10 upregulated lncRNAs and 10 downregulated lncRNAs were listed for enrichment analysis. Interestingly, we found that these lncRNA and mRNA are involved in biological process, molecular function, and cellular component in addition to various signaling pathways, suggesting that the pathogenesis of POCD involves lncRNAs and mRNAs differential expression. Consequently, the genetic dysregulation between the non-POCD and POCD patients participates in the occurrence and development of POCD, and could be served as diagnostic biomarkers and drug targets for POCD treatment. Postoperative cognitive dysfunction (POCD) is a severe central nervous system (CNS) complication in the cognitive function common in elderly patients after anesthesia and surgery. The condition involves symptoms rang from mild to serious cognitive deficits affecting life quality . POCD isLong non-coding RNAs (lncRNAs) are set of non-coding RNA with more than 200 nucleotides lengths . InteresWe selected hip and knee replacement patients as clinical cases to detect the lncRNAs and mRNAs profiles in POCD patients non-POCD patients using microarray analysis. Furthermore, Gene Ontology (GO) and KEGG analyses were performed to study the potential functions of the dysregulated lncRNAs and mRNA. Finally, network interaction of lncRNAs–mRNAs in the POCD patients was constructed.The study was approved by the ethic committee of Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University (project number KY2018001) and registered with Chinese Ethics Committee of Registering Clinical Trials in 2018 (Unique identifier ChiCTR-INR-15007607). All participating patients were informed with the aim of the study and signed the related consent forms.Two hundred patients admitted to Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University to carry out hip or knee replacement surgery have participated in this study. Patients selected based on firm criteria which were (1) age at >60 years old; (2) anesthetic procedure (American Society of Anesthesiologists Grade 1–3); and (3) mental health. It was decided to exclude patients beyond 85 years old, patients with nervous system diseases, preoperative stoke, and other central nerve system diseases cases (preoperative MMSE < 24 scores), and patients who suffered from multiple injuries and craniocerebral injury.All patients were asked to fast from food 8 h before the operation and from water 3 h before anesthesia. The anesthetic procedure was combined of spinal and epidural anesthesia at the L3–L4 intervertebral disc in a side-lying position using an 18-gage needle and 25-gage spinal needle . Then, the 2 ml mixed solution consisting of 0.5% bupivacaine  and 3.33% glucose  was slowly injected to subarachnoid space. The vital sign of patients was monitored during the surgery. All the participants were given the same analgesia doses 12 μg/kg fentanyl  and 10 mg tropisetron hydrochloride  for 2 days.For cognitive function evaluation, a battery of neuropsychological tests were followed 1 day before and 1 month after the surgery by trained physician. The tests consisted of the MMSE, Symbol Digit Modalities Test (SDMT), Digit Span Test (DST), Trail Making Test A, and Clock Drawing Test (CDT). The cognitive functions were principally assessed including memory, learning concentration, and executive function. Briefly, MMSE was designed to evaluate and quantify the global cognitive state, assess memory, spatial–temporal orientation, and reasoning . SDMT waZ score, and the participants Z ≥ 1.96 were included in POCD  value, mean variation, and baseline score were calculated to statistically validate the data . The lea in POCD .Three days after the surgery, 8 ml of peripheral blood samples were collected from the patients in pro-coagulant tube and centrifuged (at speed of 2500 rpm for 10 min). The supernatant was taken and stored in liquid nitrogen until the test. Random number table was applied to select three patients with early POCD  and three patients with non-POCD was as control . Total RNA was extracted using TRIzol reagent kit based on vendor instructions, the plasma. RNA integrity was assessed by Agilent Bioanalyzer 2100 (Agilent Technologies), and the purity was measured with NanoDrop2000 spectrophotometer (Thermo Scientific). RNA samples with OD260/280 > 1.8 and OD260/230 > 1.5 were taken for further testing.RNA samples were sent to Shanghai OE Biotech Inc.  to carry out. The sample labeling, hybridization, and washing were performed according to service provider protocols. In brief, cDNAs was synthesized for from RNA samples then synthesized cRNAs. Second cycle cDNAs were synthesized from cRNAs and followed by fragmentation and biotin labeling, the second cycle cDNAs were hybridized onto the Affymetrix Human OE lncRNA microarray , comprising 66,741 human lncRNAs and 25,986 coding mRNA. The chips were then washed, stained, and scanned by Affymetrix Scanner 3000 .Affymetrix Gene Chip Command Console  software was used to extract raw data. Expression Console  software offered RMA normalization for both gene and exon level analysis. Then, Genesrping software  was employed to carry out the basic analysis. Venn Diagram software was performed to generate high-resolution Venn plots.p-values were calculated and followed to measure pathway correlation to the conditions. Method of Figure 1A.To understand the function of lncRNAs associated with the PODC, we first established the role of differentially expressed mRNA through GO and KEGG analyses. The lncRNAs then were linked to their adjacent mRNAs to establish their potential function . PearsonP < 0.05 was viewed as statistically significant difference.In this study, SPSS software version 17.0  and GraphPad Prism 5.0  were used for data analysis and the experimental results were presented as mean values ± standard deviation (SD); Table 1. No significance variations were noticed in the scores of the all participants at first day before surgery (P > 0.05). On the other hand, CDT scores were noticeably lower in some patients and they were taken as POCD group compared with non-POCD group after 1 month of the surgery (Table 2). Whereas, the general characteristics and intraoperative conditions were statistically insignificant. One month after hip or knee replacement surgery, 21 patients (11.6%) were diagnosed as cognitive dysfunction, performed remarkably worse verbal IQ, performance IQ, and working memory compared with other non-POCD patients (Table 3).Of these 200 patients, 181 patients (91.5%) achieved the whole tests, while 19 patients (9.5%) did not complete the tests due personal reasons. The general characteristics of the patients including age, body mass index, gender, and education are presented in Figure 1B). As shown in Figures 1C,D, the scatter plot and PCA were applied to measure dissimilarities in lncRNAs expression between POCD group and control group, the detection revealed that in scatter plot and PCA, the overall distribution of the two groups of data is concentrated .Chip data dispersion was illustrated in box–whisker plot, scatter plot, and PCA. From box–whisker plot, no statistical variance was seen neither in the lncRNAs distributions nor in mRNAs expression profile in the samples  have shown the distinct lncRNAs expression profile between the groups; 68 lncRNAs were observed to be differentially expressed in the POCD patients, including 29 and 39 upregulated and downregulated, respectively (see Supplementary Tables S2). The top 10 upregulated lncRNAs were identified as lnc-FAM53B-2_1, ENST00000615844.1, lnc-MYL2-2_1, lnc-NKD2-1_1, lnc-EVC-3_1, lnc-SLC5A7-1_1, NR_024101, lnc-DCP1B-6_1, lnc-MNAT1-8_1, and lnc-TNFRSF19-4_1 . Whereas the top 10 downregulated lncRNAs were NR_024187, lnc-PTPRU-7_1, lnc-LIPC-6_1, lnc-CTD-2144E22.5.1-26_1, lnc-OTX1-7_1, ENST00000618386.1, lnc-PRL-9_1, lnc-PAG1-9_1, lnc-ZNF729-4_1, and lnc-RAP2C-5_1 . These lncRNAs were further studied by bioinformatics tools.Hierarchical clustering analysis data and heatmap . At the same time, these mRNA sequences were predicted to target different cellular components including cytoplasmic stress granule, transcriptional repressor complex, clathrin-coated vesicle, NatA complex, cullin-RING ubiquitin ligase complex, cytosolic large ribosomal subunit, Cul5-RING ubiquitin ligase complex, Cdc73/Paf1 complex, clathrin complex, condensed chromosome, and outer kinetochore . On the molecular level, the other upregulated RNAs were involved in modulating the cells by being nucleosomal DNA binding, RNA binding factors, protein deacetylase activity, poly(A), RNA polymerase II repressing factor, serine/threonine kinase, signaling receptors, interleukin-12 receptor binding factor, and 1-phosphatidylinositol-3-phosphate 4-kinase activity . On other dimension, KEGG data of the upregulated mRNAs were predicted to take part in transcriptional misregulation in cancer, Epstein–Barr virus infection, pantothenate and CoA biosynthesis, endocytosis, spliceosome, RNA polymerase, protein processing in endoplasmic reticulum, alcoholism, N-glycan biosynthesis, and Huntington’s disease .Gene Ontology analysis for the main overexpressed mRNA has revealed that these transcriptomes are involved in biological processes through series of functions including deacetylation, cytoplasmic mRNA processing, stem cell population maintenance, and positive regulation of immunity against viruses by vesicle organization, regulation of mitotic cell cycle, peptidyl-threonine phosphorylation, endocytosis, histone deacetylation, and actin cytoskeleton reorganization . Whereas the highest 10 cellular components related to the downregulated mRNAs were cytoplasmic stress granule, Cajal body, condensin complex, cullin-RING ubiquitin ligase complex, cytosolic large ribosomal subunit, Cul5-RING ubiquitin ligase complex, Cdc73/Paf1 complex, nucleoplasm, outer dense fiber, and condensed chromosome outer kinetochore . Similarly, the top 10 functions on molecular mechanism include poly(A) RNA binding, nucleosomal DNA binding, signaling protein receptor, serine/threonine kinase, phosphatase regulator, SUMO-specific protease activity, protein serine/threonine kinase activity, ATP binding, RNA polymerase II intronic transcription regulator, neuropeptide Y receptor activity, and structural constituent of ribosome . Laterally, the top 10 affected pathways of mRNA sequences downregulated were Epstein–Barr virus infection, pantothenate and CoA biosynthesis, spliceosome, RNA polymerase, Protein processing in endoplasmic reticulum, transcriptional misregulation in cancer, alcoholism, N-Glycan biosynthesis, carcinogenesis viral RNA, and adipocytes lipolysis regulation .On the basis of the enrichment scores, the top 10 biological processes targeted by downregulated mRNAs were identified as cytoplasmic mRNA processing body assembly, snRNA transcription, polymerase II promoter, cell division, regulation of mitotic cell cycle, outflow tract morphogenesis, stem cell population maintenance, regulation of translation, regulation of fertilization, meiotic chromosome condensation, and regulation of myotube differentiation (trans (trans-prediction) and Venn analyses, the lncRNA–mRNA interaction network was constructed. The data showed that the 10 upregulated lncRNAs were correlated with amounts of mRNAs through trans analysis. The lnc-FAM53B-2_1 in particular was the highest upregulated lncRNA. Interestingly, it interacted with the majority of differentially dysregulated mRNA sequences. It was also noticed that SMARCE1, ATXN2, SMC4, and IL23R were remarkably unregulated in POCD patients . Venn diagram showed that the highest five upregulated lncRNAs were lnc-FAM53B-2_1, ENST00000615844.1, lnc-MYL2-2_1, lnc-NKD2-1_1, and lnc-EVC-3_1. While the main overexpressed mRNAs were AC009487.4, FANK1, OLFML2B, OXSR1, RNU6-18P, RP11-483E7.1, SMARCE1, and ZFR .Based on the information of Figure 6A). Venn diagram also showed that the main five downregulated lncRNAs, were NR_024187, lnc-PTPRU-7_1, lnc-LIPC-6_1, lnc-CTD-2144E22.5.1-26_1, and lnc-OTX1-7_1 and they were slightly correlated with RP5-834N19.1 and PARL mRNA .In addition, the top 10 downregulated lncRNAs were also associated with many mRNA sequences. As the most downregulated lncRNA in POCD, NR_024187 was correlated with that of NEK3, XKRX, VNN1, TBXAS1, and HDAC5 (P < 0.05). Among them, 39 mRNAs were upregulated and 76 were downregulated (P < 0.05). Variations in mRNA expression levels of the POCD and non-POCD group are illustrated in Figure 7.Microarray data analysis also showed the overexpressed genes. Total of 115 mRNA sequences were dysregulated between the POCD and control groups (FC > 2, Postoperative cognitive dysfunction is a postoperative complication with high incidence and poor prognosis . At presAs a class of important ncRNA, lncRNA plays vital role in central system . In receIn the present study, various neuropsychological tests were adopted to evaluate cognitive functions 1 day before and 30 days after knee or hip replacement surgery to distinguish between POCD and non-POCD patients. Then, lncRNA sequences were studied from all the patients using developed microarray chips. Microarray analyses revealed that there were total of 29 upregulated lncRNAs and 39 mRNAs, 39 downregulated lncRNAs, and 76 mRNAs associated with POCD. Likewise, the GO and KEGG were performed to predict the roles of the dysregulated lncRNAs, and the co-expression mRNA. Furthermore, network interaction of lncRNAs and mRNAs in the POCD patients was constructed to explore the relationship between lncRNAs and mRNAs with the disease. As POCD symptoms are memory decline, poor attention, learning disability, and deficit function execution. Therefore, this experiment uses a series of neuropsychology tests to assess these five categories of cognition functions to distinguish whether cognitive impairment existed before surgery it has occurred after the surgery. To increase the accuracy, all cognitive functions were measured by more than one test to diagnose the POCD patients.Bioinformatics analyses were aimed to indicate the potential functions of the dysregulated lncRNAs that have been identified in our study. GO analysis has illuminated the functions of genes through biological process, cellular component, and molecular function. While KEGG is normally used to study the possible roles of expressed RNA with correlation to cell signaling pathways .In our analysis of biological process, it was noticed that the differentially expressed lncRNAs, mainly participated in process of protein and histone deacetylation, cytoplasmic mRNA processing body assembly, regulation of cell cycle, and stem cell population. Previous study has also pointed out the role of histone acetylation/deacetylation in neurodegenerative conditions . In concFurthermore, pathway analysis revealed that there were 10 pathways implicated in POCD patients, Epstein–Barr virus infection, and pantothenate and CoA biosynthesis were the highly enriched pathways in POCD patients. Epstein–Barr virus, a human gamma-herpesvirus, potentially facilitates the damage of autoimmunity and CNS tissue, causing psychiatric symptoms and cognitive dysfunction . There aUntil the present, the functions of lncRNAs are not entirely clear. The co-expression network of aberrantly expressed mRNAs and lncRNAs in POCD was constructed to further understand of the role of lncRNAs via calculation and inference.In the present study, we have selected the top 10 up and downregulated lncRNAs to construct the lncRNA-mRNA co-expression network of interactions to predict the genes targeted by lncRNA. We found that the increased lncRNA, lnc-FAM53B-2_1, correlated with that of SMARCE1, ATXN2, SMC4, and IL23R, while main reduced lncRNA was NR_024187 and has influence on NEK3, XKRX, VNN1, TBXAS1, and HDAC5. Previous study showed SMARCE1 to be associated with the occurrence and development of non-syndromic intellectual disability . IncreasHowever, it will be worthwhile to evaluated neurological function in long-term manner in POCD patients. In addition, more hands on researches should be conducted to investigate the exact mechanisms and function of genes that have been reported in this study to examine the specific interaction mechanisms between lncRNAs and POCD.We have recorded that lncRNAs and mRNAs in serum were differentially expressed in patients with POCD compared with patients without POCD after hip or knee replacement surgery. These vital findings may contribute to well understand the mechanism of POCD and provide new biomarkers for diagnosis and monitoring the progression of POCD and potential therapeutic target for POCD treatment.F-XY and YZ conceived and designed the experiments. YZ, Y-XL, Q-XX, and QL performed the experiments. Y-XL, RD, ID, and JB analyzed the data. YZ, MA-H, and Y-XL wrote the manuscript. MA-H and ID participated in improving the paper and making the language more authentic during the revision.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.


Recent evidence illustrates that Kir4.1 channels play important roles in bringing about the actions of antidepressant drugs and modulating BDNF expression in astrocytes. Although the precise mechanisms remain to be clarified, it seems likely that inhibition (down-regulation or blockade) of astrocytic Kir4.1 channels attenuates K+ buffering, increases neuronal excitability by elevating extracellular K+ and glutamate, and facilitates BDNF expression. Conversely, activation (up-regulation or opening) of Kir4.1 channels reduces neuronal excitability by lowering extracellular K+ and glutamate, and attenuates BDNF expression. Particularly, the former pathophysiological alterations seem to be important in epileptogenesis and pain sensitization, and the latter in the pathogenesis of depressive disorders. In this article, we review the functions of Kir4.1 channels, with a focus on their regulation of spatial K+ buffering and BDNF expression in astrocytes, and discuss the role of the astrocytic Kir4.1-BDNF system in modulating CNS disorders.Brain-derived neurotrophic factor (BDNF) is a key molecule essential for neural plasticity and development, and is implicated in the pathophysiology of various central nervous system (CNS) disorders. It is now documented that BDNF is synthesized not only in neurons, but also in astrocytes which actively regulate neuronal activities by forming tripartite synapses. Inwardly rectifying potassium (Kir) channel subunit Kir4.1, which is specifically expressed in astrocytes, constructs Kir4.1 and Kir4.1/5.1 channels, and mediates the spatial potassium (K After screening CNS drugs in Kir4.1-expressing HEK293 cells, we found that several antidepressant agents reversibly inhibited Kir4.1 channels in concentration- and subunit-dependent manners. Of these, tricyclic antidepressants (TCAs), amitriptyline, nortriptyline, desipramine, and imipramine, inhibited Kir4.1 channel activities in a voltage-dependent manner ,31. In aBesides antidepressant drugs, several agents have been shown to interact with Kir4.1 channels. These include anti-malarial drugs, quinacrine and chloroquine ,35, an i2+ signaling and multiple second messenger-protein kinase cascades) and secrete various neuroactive molecules including BDNF o o , these ed [Glu]o ,49,50, i+ channels , the activation of GABAergic neurotransmission by inhibiting GABA transaminase  or stimulating GABAA receptors , the blockade of voltage-gated Ca2+ channels  or the antagonism of glutamate receptors  . Ki. Ki+ bufsyndrome ,56. Patil domain ,57,58. Ad [Glu]o . Seizurepocampus  60,61].,61.+ bufgenesis) . The deagenesis)  seem to  tubules ,56.+ buffering action of astrocytes and caused hyperexcitation of amygdala neurons in NER, which led to induction of generalized tonic-clonic seizures. In humans, several clinical studies showed the down-regulation and impaired functioning of Kir4.1 channels in patients with TLE o and [Glu]o levels and facilitates BDNF expression, which seem to improve DDs  demonstrated that astrocytic Kir4.1 channels in the lateral habenula closely regulate the expression of neuronal bursting and depressive behaviors in rats o and [Glu]o levels and attenuates BDNF expression. This seems to be involved in the stress-induced atrophy of the limbic structures and the development of DDs. Since BDNF plays a key role in neural plasticity, development, and cell survival by facilitating synaptogenesis, neural sprouting, neurogenesis, and reactive gliosis, the astrocytic Kir4.1-BDNF system is expected to serve as a novel target for the treatment of CNS disorders.Expression of BDNF in astrocytes is now receiving a lot of attention as an important modulator of CNS disorders. Particularly, astrocytic Kir4.1 channels seem to play a crucial role in modulating BDNF expression. Evidence is accumulating that inhibition of Kir4.1 channels attenuates K