An anuric peritoneal dialysis patient with diabetes mellitus, congestive heart failure, and anasarca developed severe hyperglycemia with hypertonicity causing profound neurological manifestations after prolonged and continuous use of hypertonic (4.25%) dextrose dialysate. She expired with hypotensive shock from a new myocardial infarction soon after completion of treatment with insulin infusion. The degree of the presenting hypertonicity far exceeded the value expected from the degree of hyperglycemia. We identified prolonged peritoneal dialysis with hypertonic solutions and profound extracellular volume expansion as the causes of the excessive hypertonicity. Hyperglycemia developing in diabetic patients treated for anasarca by peritoneal dialysis after continuous use of hypertonic dextrose dialysate is associated with the risk of excessive hypertonicity with severe clinical manifestations. Hyperglycemic crises developing in patients with preserved renal function create two categories of diagnostic and treatment challenges. The first category of challenges includes identification and management of the clinical entity – or entities – which caused the hyperglycemia. The second category of challenges consists of body fluid disturbances that can be life-threatening. Major hyperglycemic body fluid disturbances causing severe clinical manifestations and requiring prompt management include extracellular volume deficits, hypertonicity, potassium deficits and maldistribution between the intracellular and extracellular compartments, and acid-base disturbances, mainly metabolic acidosis -4. OtherAbsence or severe reduction of renal function does not diminish the need for identification and management of the causes of hyperglycemia, but modifies substantially its biochemical and clinical manifestations. Routinely patients on chronic dialysis with severe, or even extreme, hyperglycemia are asymptomatic  or exhibWe report a patient on continuous peritoneal dialysis presenting with extreme hyperglycemia, pulmonary edema, and severe hypertonicity resulting in dire clinical consequences. This case report illustrates conditions, other than the degree of hyperglycemia, that aggravate the hyperglycemia-induced hypertonicity in oligo-anuric patients.An anuric 60-year-old woman with diabetic nephropathy had been treated for four months by continuous ambulatory peritoneal dialysis (CAPD) with four daily exchanges and 2.0 L fill volume. She had a history of coronary artery disease with an anterior myocardial infarction seven months prior to the start of CAPD, followed by severe congestive heart failure with an ejection fraction of 23%. Her dry weight, documented one month after the start of CAPD by absence of both edema and hypertension, was 41.2 kg. Her height was 149 cm. She had not been seen by her CAPD providers for three months because of transportation difficulties when she was admitted with grand mal seizures followed by coma. One week prior to this admission, she communicated by telephone call with one provider. At that time, she reported total body swelling, moderate dyspnea and a body weight exceeding 70 kg. She was advised to use exclusively hypertonic dextrose (4.25%) CAPD dialysate and add two exchanges per day with short (1 hour) dwell times. Information obtained from relatives revealed that in the two days prior to this admission she had omitted her insulin dose because of anorexia and greatly decreased food intake.On admission, she was comatose with a blood pressure of 180/74 mm Hg, regular pulse rhythm with a rate of 110 beats per minute, and temperature of 36.5 degrees Celsius. Her weight was 63.6 kg. Remarkable findings on physical examination included anasarca detected even in her scalp, a ventricular gallop, and crackles in both lung fields. Chest X-ray confirmed the presence of pulmonary edema and electrocardiogram was unchanged from recent tracings, which were consistent with old transmural myocardial infarction. Laboratory tests on admission revealed extreme hyperglycemia, severe hypertonicity, hypokalemia and respiratory acidosis.Over the next 30 hours, she received insulin boluses and had frequent (every 2-4 hours) determinations of serum glucose and potassium concentrations. Serum glucose levels decreased progressively, while serum potassium levels remained between 3.1 and 3.4 mmol/L without administration of potassium salts. Blood pressure progressively decreased during treatment. At the end of treatment, she developed frank hypotension (blood pressure 80/60 mm Hg) and increased tachycardia to a pulse rate of 125 mm Hg, while remaining comatose. Chest X-ray revealed worsening pulmonary edema, while electrocardiogram was consistent with a new myocardial infarction and serum levels of cardiac enzymes  were substantially elevated. Subsequently, she developed worsening hypotension and expired within three hours. Table Serum tonicity (effective osmolarity), calculated as 2x(Serum sodium) + Serum glucose in mmol/L, is consistently elevated in hyperglycemia . This caHypertonicity in hyperglycemic episodes developing in oligo-anuric patients is routinely modest because its only component is the glucose gain. Loss of hypotonic fluid does not usually occur in these episodes. Furthermore, hyperglycemic hypertonicity is modulated by thirst and water intake and retention in approximately one-third of the patients on dialysis . In a reWhen the only source of hypertonicity is extracellular glucose gain, serum sodium concentration is invariably depressed because of the exit of intracellular water into the extracellular compartment in hyperglycemic episodes , 5-9. LoWe identified prolonged peritoneal dialysis with hypertonic dialysate as one cause of the severe hypertonicity in the patient presented in this report because of the elevated serum sodium concentration at presentation with extreme hyperglycemia. Loss of water via peritoneal ultrafiltration in relative excess of losses of sodium and potassium was probably aggravated by the two daily exchanges with short dwell times. Early after intraperitoneal instillation of hypertonic dialysate, sodium sieving results in lower sodium concentration in the dialysate than in plasma. The lowest dialysate sodium concentration is observed approximately one hour after intraperitoneal infusion of the dialysate .A second component of the excessive hypertonicity of our patient at presentation with hyperglycemia was the presence of anasarca. The influence of anasarca on the degree of hypertonicity was identified by analyzing the rates of increase in serum sodium and decline in tonicity during treatment. Throughout treatment, the patient received no fluids and had no detectable fluid losses. Under these conditions, serum sodium concentration increases through osmotic transfer of extracellular fluid into the intracellular compartment as serum glucose concentration decreases , 9. We cThe change in tonicity (ΔTon) during development or correction of hyperglycemia is the difference between the changes in the osmotic contributions of the changes in serum glucose (ΔGlucose) and sodium concentrations (ΔNa), or ΔTon = ΔGlucose – 2xΔNa . During The reason for the low rate of change in serum sodium concentration and high rate of change in tonicity during treatment of the hyperglycemic episode in our patient was the profound expansion of extracellular volume. The main determinants of the changes in tonicity in hyperglycemic episodes occurring in oligo-anuric patients without losses of hypotonic fluids are the degree of hyperglycemia and the state of extracellular volume . For theTotal body water at our patient's dry weight calculated by the Watson formula  was 23.9Two last points of interest in this case report are the evolution of serum potassium concentration during treatment and the presenting acid-base disturbance. A decrease in serum potassium concentration during treatment is routinely observed during treatment of anuric hyperglycemia as a result of direct action of insulin on cell membranes and correction of hypertonicity . InfusioSymptomatic hypertonicity from severe hyperglycemia without hyponatremia in oligo-anuric patients calls for search for conditions causing loss of hypotonic body fluids. Presence of pronounced edema is another risk factor for excessive hypertonicity in oligo-anuric patients developing severe hyperglycemia. Peritoneal dialysis in diabetic patients with the exclusive use of hypertonic solutions is associated with significant risks for hyperglycemia and hypertonicity, especially in the presence of pronounced edema, and should be performed in a hospital setting with frequent monitoring of the clinical status of the patient and of serum glucose, sodium, and potassium concentrations.


Gastroesophageal reflux disease (GERD) can cause several upper airway symptoms and alter the physiology of nasopharyngeal mucosa, while upper airway diseases in turn might also exacerbate GERD symptoms. For a long time, asthma was considered a risk factor of GERD in the literature. Asthma and allergic rhinitis (AR) are usually identified as united airway disease according to similar epidemiology and pathophysiology; however, the association between AR and GERD is less elucidated. We aimed to evaluate whether AR would increase the development of GERD. Patients diagnosed as AR were identified from the National Health Insurance Research Database between January 1, 2000 and December 31, 2005 without prior history of gastroesophageal reflux disease. The outcome of interest was new-onset GERD. Cox regression models were applied to calculate the hazard ratio (HR) of GERD. We analyzed the data of 193,810 AR patients aged 18 years or older and being free of AR at baseline. The AR cohort  had a significantly increased risk of GERD over a non-AR cohort (n = 96905) (adjusted HR (aHR) 1.94; 95% CI = 1.88–1.99, p < 0.001). AR may have stronger correlation with GERD than does asthma, although asthma might increase GERD risk by means of certain pathways shared with AR. It is defined by its troublesome symptoms and/or complications caused by the refluxed contents from the stomach, creating a great adverse impact on health and quality of life as well as consuming financial resources in medical insurance2. The typical symptoms of GERD are regurgitation and heartburn. In addition to cardinal symptoms, reflux from stomach also induces so-called atypical or extra-esophageal symptoms, including cough, hoarseness, pharyngitis, laryngitis, chest pain, sinusitis, otitis media, dental erosions and sleep disturbance. Since there are many factors contributing to the development of reflux and reflux-related symptoms, GERD is identified as a multi-factorial disease. Even today, the pathophysiology of GERD is not fully understood and many mechanisms have been proposed to explain the development of GERD, inclusive of obesity, transient lower-esophageal sphincter relaxations (TLESRs), hiatus hernia, acid pockets, visceral hypersensitivity, impaired esophageal mucosal integrity, poor esophageal clearance, and delayed gastric emptying5.Gastro-esophageal reflux disease (GERD) is one of the most prevalent gastrointestinal diseases and its prevalence is increasing worldwide6. Asthma is a complex condition characterized by variable and recurrent symptoms as the result of reversible airflow obstruction, bronchial hyper-responsiveness, and chronic airway inflammation. In a meta-analysis, asthma prevalence is 4.6% in patients with GERD and 3.9% in controls, while GERD symptom prevalence is 59.2% in patients with asthma and 38.1% in controls. Odds ratio is 2.26 for asthma in GERD, while it is 5.45 for GERD in asthma7. Cough and increased respiratory effort might induce reflux in patients with asthma8. On the other hand, exposure to acid content reflux may exacerbate asthma as a result of direct pulmonary tree damage as well as bronchoconstriction indirectly via vagal stimulation9. Asthma and allergic rhinitis (AR) are common airway diseases and are viewed as a united airway disease. They frequently occur together and share similar physiological traits, including heightened reactivity to a variety of stimuli and heightened bronchial hyper-responsiveness10.In the literature and clinical practice, certain diseases are observed to be associated with GERD. Asthma is the most studied disease in its relationship with GERD12. Sneezing, nasal congestion, nose/throat itching, and anterior/posterior rhinorrhea are the four cardinal symptoms of AR. The immunopathology of AR and asthma are quite similar in terms of cellular influx of mast cells, eosinophils, and T-helper type 2(Th2) cells. In addition, similar mediators can also be found in patients with AR and asthma, such as histamine, cysteinyl leukotrienes, chemokines, and Th2 cytokines10.AR is defined as an inflammatory process in the nasal mucosa that is stimulated by exposure to allergens. AR is very common, affecting 9 to 42% of children and 10 to 30% of adultsAs above, there is evidence of an association between asthma and GERD; however, few studies have evaluated the impact of AR on GERD. The aim of this study, which used the Taiwan National Health Insurance Research Database (NHIRD) database, was to access the relationship of AR with newly developed GERD in adults. However, we used ICD-9 codes of diagnosis for patient selection, as it is difficult to validate the accuracy of the diagnosis set by the treating doctor.From Jan 1, 2000 to Dec 31, 2005, we conducted a study with 96,905 and 96,905 children as AR cohort and non-AR cohort controls respectively from LHID2005. The mean age of the AR cohort was 43.72 ± 16.9 years. The distribution of age, gender, and comorbidities were similar between AR cohort and non-AR cohort groups , gender , diabetes, hyperlipidemia , atopic dermatitis , chronic sinusitis , rhinitis , and depression  , a subgroup of GERD24. However, the role of allergy in chronic rhinosinusitis with and without nasal polyps has remained controversial25. Anaphylaxis is a serious allergic reaction that is rapid in onset and typically presents many different symptoms over minutes and hours. The most common symptoms included an itching rash, throat or tongue swelling, vomiting, shortness of breath and low blood pressure. It is caused by the release of inflammatory mediators and cytokines from mast cells and basophils, typically due to an immunological reaction26. Since anaphylaxis is a short-term immunity response, it has less impact on the chronic development of GERD. Additionally, there was too small a sample size of anaphylaxis incidences in our cohort.In our study, we also evaluated the risk factors of GERD in AR patients Table . Older aOur studies had several limitations in that we did not consider confounding by lifestyle, obesity, smoking and alcohol consumption. Moreover, the diagnosis of AR and GERD was according to ICD-9 code. The tools for exact diagnosis of AR and GERD might be dissimilar, thus under-diagnosis or over-diagnosis might lead to inaccurate interpretation. Besides, the evaluation of the associated risks and diseases was also according to ICD-9 code, and the misclassification of ICD-9 could lead both to under- and overestimates of the diseases and the risks. Using a register-based data on clinical visits for allergic rhinitis and GERD means that a large number of subjects in the population suffering from allergic rhinitis and/or GERD would not be included in the study. Some patients with milder symptoms might self-medicate instead of seeking medical care. It is therefore likely that our study includes the more severe cases of allergic rhinitis and GERD. On the other hand, the peak onset of AR is around 20 years of age. In our study, the mean diagnosis age of AR was 44 years, but not the onset age; the reason might be that many younger patients prefer to self-medicate; thus, the exact period of diagnosis of GERD after onset of AR might well be longer.In conclusion, our results provide evidence that AR, especially with asthma, has an increased risk factor for developing new-onset GERD in adults. Asthma might increase the risk of GERD by means of certain mechanisms shared with AR; however, the precise interaction and causal relationship between AR, asthma, and GERD is worth further investigation.The study database used claims data from the National Health Insurance Research Database (NHIRD), which is managed by the Taiwan National Health Research Institute (NHRI). The NHIRD contains comprehensive healthcare information for virtually all of Taiwan’s nationally insured population, including demographic data, clinic visit date, diagnostic codes, and prescription details. For the study, we used a subset of NHIRD known as the Longitudinal Health Insurance Database (LHID) 2005, which contains ambulatory and inpatient medical care records for one million randomly sampled subjects enrolled into the NHI system in the year of 2005. According to the NHRI, there were no statistically significant differences in age, gender, or healthcare costs between the LHID 2005 and NHIRD.We identified allergic rhinitis  between January 1, 2000 and December 31, 2005 for the AR cohort. The AR cohort group was compared with a comparison cohort, which consisted of patients who had never been diagnosed as having allergic rhinitis, matched (1:1) on the basis of age, sex, comorbidities, propensity score and index year. The date of first diagnosis of allergic rhinitis for each patient was defined as the index date. Subjects were excluded if they were less than 18 years of age, had gastroesophageal reflux disease (GERD) diagnosed before the index date, or had incomplete follow-up insurance data. The protocol was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-F(I)-20150168). All methods were performed in accordance with the relevant guidelines and regulations.The outcome of interest was a diagnosis of ICD-9-CM 530.11 ). Patients were followed from the index date to the new onset of gastroesophageal reflux disease, death, disenrollment from the national health insurance, or the end of the study date , whichever came first.Inpatient and outpatient files from the year prior to the index date were used to obtain information on co-morbidities including atopic dermatitis (ICD-9 code: 691.8), chronic sinusitis (ICD-9 code: 472.0), rhinitis , and nasal polyps (ICD-9 code: 471) were evaluated.t-test evaluated differences of two cohorts in continuous variables. Cox proportional-hazards regression analysis was performed to examine the risk of GERD in the AR cohort compared with the non-AR cohort during the follow-up period. Several co-variables, including age, gender, and co-morbidities, were adopted in the statistical analysis model. Hazard ratios (HR) and 95% confidence intervals (CI), using non-AR cohort without asthma history as the reference, were calculated to show the risk of GERD in the interaction analysis between AR and asthma. A Kaplan–Meier curve was used to estimate the probability of GERD, and the log-rank test was used to evaluate the differences between cohorts. All statistical operations were performed using the SAS 9.3 statistical package; all P-values were 2-sided, and P-values < 0.05 were considered significant.Pearson chi-square test or Fisher’s exact test was used to evaluate differences in categorical variables between AR and non-AR cohorts. Independent


Increasing evidence has indicated that long noncoding RNAs (lncRNAs) are crucial regulators affecting the progression of human cancers. Recently, lncRNA downregulated in liver cancer stem cells (lnc-DILC) was identified to function as a tumor suppressor inhibiting the tumorigenesis and metastasis in liver cancer and colorectal cancer. However, to date, little is known about the functional roles of lnc-DILC in modulating malignant phenotypes of clear cell renal cell carcinoma (ccRCC) cells.lnc-DILC expression in human ccRCC tissues was detected by qRT-PCR. Overexpression and knockdown experiments were carried out to determine the effects of lnc-DILC on ccRCC cell proliferation, migration and invasion. To reveal the underlying mechanisms of lnc-DILC functions in ccRCC cells. RNA immunoprecipitation, RNA pull-down, in vivo ubiquitination, co-immunoprecipitation and western blot assays were performed.Here, we identified that lnc-DILC levels were dramatically downregulated in ccRCC tissues. Loss of lnc-DILC expression was correlated with larger tumor size, advanced tumor grade and lymph node metastasis, and also predicted worse prognosis in patients with ccRCC. Functionally, knockdown and overexpression experiments demonstrated that lnc-DILC inhibited cell proliferation, migration and invasion in ccRCC cells. Mechanistic investigation revealed that lnc-DILC bound to tumor suppressor PTEN and suppressed its degradation. lnc-DILC repressed the PTEN ubiquitination through blocking the interaction between PTEN and E3 ubiquitin ligase WWP2 and recruiting the deubiquitinase USP11 to PTEN. Moreover, we demonstrated that PTEN–AKT signaling was crucial for lnc-DILC-mediated suppressive effects.In summary, our research revealed a novel mechanism by which lnc-DILC regulates PTEN stability via WWP2 and USP11, and shed light on potential therapeutic strategies by the restoration of lnc-DILC expression in patients with ccRCC. Renal cell carcinoma (RCC) originates from renal tubular epithelial cells and is one of the most frequent cancers of the urinary system . RCC canLong noncoding RNAs (lncRNAs) are a group of transcripts more than 200 nucleotides in length and not capable to translated into proteins. LncRNAs were previously considered as “transcriptional junk” . HoweverLnc-DILC (lncRNA downregulated in liver cancer stem cells), a newly identified lncRNA, locates at the chromosomal locus 13p34. In liver cancer and colorectal cancer, lnc-DILC acts as a tumor suppressor to inhibit the tumorigenesis and metastasis. Lnc-DILC was found to suppress the IL-6/STAT3 signaling via inactivating IL-6 transcription , 22. Con68 pairs of ccRCC and matched normal tissue samples were collected from ccRCC patients who underwent surgery at the Luoyang Central Hospital. None of patients received other therapy before surgery, such as chemotherapy, radiotherapy and targeted therapy. Written consent was obtained from all patients. This study was approved by the Institutional Ethics Committee of the Luoyang Central Hospital.The human ccRCC cell lines, ACHN, 786-O, 769-P, Caki-1 and OS-RC-2, and a normal human renal cell line HK-2 were purchased from the Cell Resources Center of Chinese Academy of Sciences. All cells were cultured in RPMI-1640 medium (Gibco) containing 10% FBS (Gibco) at 37 °C. siRNAs targeting lnc-DILC or PTEN was purchased from Ribobio Company . Full-length lnc-DILC or PTEN was synthesized by GENECHEM Company  and subcloned into the pcDNA3.1 vector. Above siRNAs or plasmid were transfected into ccRCC cells by using Lipofectamine 2000 (Invitrogen) according to the the manufacturer’s instructions. 48 h after transfection, the cells were subjected to further experiments.−△△Ct methods. The primer sequences were shown as follows: lnc-DILC-sense: TGGCCTACTCCCAAGAAGATA, lnc-DILC-antisense: CATCCACAGCACGTCCTAAT; PTEN-sense: CCCACCACAGCTAGAACTTATC, PTEN-antisense: TCGTCCCTTTCCAGCTTTAC; GAPDH-sense: GGTGTGAACCATGAGAAGTATGA, GAPDH-antisense: GAGTCCTTCCACGATACCAAAG.Total RNA from tissue samples and cells were isolated by Trizol reagent (Invitrogen). The cDNA was synthesized by using EasyScript First-Strand cDNA Synthesis SuperMix Kit, and then detected by qRT-PCR using TransStart Green qPCR SuperMix . The relative expression of indicated genes was normalized to GAPDH mRNA level and calculated using 2Cell proliferation was assessed by CCK-8 (Dojindo) and clone formation assay according to the manufacturer’s instructions.The cell apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit (Sigma) according to the manufacturer’s instructions. For cell cycle analysis, cells were collected and fixed in 70% ethanol at 4 °C overnight and then incubated witi PI staining solution. Data were analyzed on a flow cytometer.5 cells suspended in 200 μl serum-free RPMI-1640 medium were plated in the upper chambers, while 500 μl RPMI-1640 medium supplemented with 10% FBS was added to the bottom chamber. 24 h later, the cells on the lower surface were fixed, stained and counted.Cell migration and invasion was examined with Transwell chambers (Corning) that coated without or with Matrigel (BD Biosciences), respectively. 1 × 10Protein samples from cells were extracted by RIPA buffer  and separated in SDS-PAGE gels and then transferred to PVDF membranes (Millipore). The membranes were incubated with primary antibodies overnight and then incubated with corresponding secondary antibodies. Protein expression was measured by enhanced chemiluminescence (Millipore).Caspase-3 activity was detected using the Caspase-3 activity assay kit  according to the manufacturer’s instructions.Cell lysates were incubated with 3 μg PTEN antibody (Abcam) and Dynabeads Protein G (Invitrogen) on the rotating platform at 4 °C overnight. After wash, the immunoprecipitated protein was subjected to western blot analysis.To test the ubiquitination level of PTEN, cells were pre-treated with 10 mM MG132 for 8 h. Cell lysates were incubated with 3 μg PTEN antibody (Abcam) and Dynabeads Protein G (Invitrogen) on the rotating platform at 4 °C overnight. After wash, the immunoprecipitated protein was subjected to western blot analysis using anti-Ubiquitin antibody .RIP and RNA pull-down assay was carried out using EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millopore) and Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher) according to the the manufacturer’s instructions, respectively.All experiments were repeated at least three times. All statistical analyses were performed using SPSS 19.0. Student t-test or one way ANOVA test was used to assess the differences between groups. The correlation between lnc-DILC expression and clinicopathological parameters was analyzed by Chi-square test. The Kaplan–Meier method and log-rank test was used to evaluate the relationship between lnc-DILC expression and overall survival time. p value less than 0.05 were considered significant.We first determined the expression pattern of lnc-DILC in ccRCC. The qRT-PCR assay was carried out to examine the lnc-DILC levels in 68 pairs of ccRCC and matched normal tissue samples. As shown in Fig. To explore the biological function of lnc-DILC in ccRCC progression, the lnc-DILC expression in five ccRCC cell lines  was assessed using qRT-PCR assay  levels when treated by PTEN siRNA or SC79 in lnc-DILC-overexpressed ACHN cell.


However, the molecular mechanism by which the sodium ion influences the recognition of the A2A AR agonists is not yet fully understood. In this study, the supervised molecular dynamics (SuMD) technique was exploited to analyse the sodium ion recognition mechanism and how its presence influences the binding of the endogenous agonist adenosine. Due to a higher degree of flexibility of the receptor extracellular (EC) vestibule, we propose the sodium-bound A2A AR as less efficient in stabilizing the adenosine during the different steps of binding.One of the most intriguing findings highlighted from G protein-coupled receptor (GPCR) crystallography is the presence, in many members of class A, of a partially hydrated sodium ion in the middle of the seven transmembrane helices (7TM) bundle. In particular, the human adenosine A The human genome encodes more than 800 different G protein-coupled receptors (GPCRs), membrane proteins characterized by a distinctive seven transmembrane helices (7TM) architecture. This superfamily of receptors recognizes an enormous variety of extracellular signals  and transmits the chemical information into the intracellular compartment, modulating many cellular activities ,2,3. Thi1, A2A, A2B, and A3, all involved in purinergic signaling . Th. Th2A AR residue . Althoug similar , the rec similar . On the  sites ) .2A AR, an unbiased MD simulation was performed from a SuMD replicate’s final state . As expected, in about 600 ns, a spontaneous unbinding event from the allosteric site was sampled . On the other hand, Na+–A2A AR complexes can trigger on the binding mechanism of the endogenous agonist, adenosine, further SuMD replicates were carried out and the results will be described in the next sections.To investigate the possible effects that these two different Na2A AR coordinates in the intermediate-active conformation (PDB ID 2YDO). We define “productive” as a trajectory that resulted in the adenosine reaching the orthosteric site. The seven productive SuMD simulations were extended for a further 100 ns of unbiased MD simulation to evaluate the stability of the bound states sampled.Ten SuMD replicates  were perECL2, as well as hydrophobic contacts with M1745.35 and transient hydrogen bonds with residues at the ECL3 .The seven productive SuMD simulations  allowed de chain C, due to2A AR conformational states, SuMD was performed on the inactive conformation of the receptor (PDB ID 4EIY), retaining the sodium in its allosteric site  the trajectories were prolonged for 100 ns (unbiased MD). As reported in + coordination within the allosteric TMD allows for the discrimination of the two main conformational states of A2A AR ; it is capable of recognizing adenosine with antithetical efficiency, as suggested by the divergent binding frequencies sampled through the SuMD simulations. As highlighted in In a schematic way, Na2A AR binding site residues were graphically depicted, using polar diagrams. As reported in To decipher the molecular basis underneath such misleading outcomes described by the SuMD simulations , cumulative maps of the interatomic contacts between adenosine and A2A AR states is fundamental to interpreting the discrepant agonist recognition pathways. In a recent computational investigation, increased flexibility of A2A AR EC domains was described in the receptor inactive state, a phenomenon that is less relevant in the active conformation and thus could help in differentiating agonist binding mechanisms [2A AR binding site on both crystallographic structures taken under examination were quite similar (~250 Å3), only a few ns of the simulation were required to reveal the different evolutions of the two systems.Deciphering the dynamics of the Achanisms . To veri2A AR, adenosine approached the receptor, interacting with the vestibular region ECL2  was exploited to perform most of the general molecular modelling operations, such as proteins and ligands preparation . All the2A AR were retrieved from the RCSB Protein Data Bank database (PDB ID 2YDO and 4EIY respectively) and handled by means of the MOE protein structure preparation tool [2A AR apo forms were obtained by simply deleting the orthosteric ligands from their respective complexes. Adenosine force field parameters were retrieved from the Paramchem web service, in concordance with CGenFF [Agonist and antagonist-bound complexes of Aion tool ,30. Hydrion tool . In the ion tool . A2A AR h CGenFF ,38.+ atoms and 111 Cl+ counterions to a final concentration of 0.154 M (A2A AR net charge was +11 for both the system-simulated 2YDO/4EIY). Equilibration was performed through a three-step procedure. In the first step, 1500 conjugate-gradient minimization steps were applied to reduce the clashes between proteins and lipids. Then, a 5 ns long MD simulation was performed in the NPT ensemble, with a positional constraint of 1 kcal mol−1 Å−2 on ligand, protein, and lipid phosphorus atoms. During the second stage, 10 ns of MD simulation in the NPT ensemble were performed constraining all the protein and ligand atoms but leaving POPC residues free to diffuse in the bilayer. In the last equilibration stage, positional constraints were applied only to the ligand and protein backbone alpha carbons for a further 5 ns of MD simulation.Systems were embedded in a 1-palmitoyl-2oleyl-sn-glycerol-3-phospho-choline (POPC) lipid bilayer, according to the pre-orientation provided by the Orientations of Proteins in Membrane (OPM) database and by using the VMD membrane builder plugin ,41. Lipi−1; the M-SHAKE algorithm constrained the bond lengths involving hydrogen atoms [All the MD simulations were performed using the following protocols: an integration time step of 2 fs; a Berendsen barostat maintained the system pressure at 1 atm; a Langevin thermostat maintained the temperature at 310 K with a low dumping of 1 psen atoms ,45,46.+ monovalent ion, as well to simulate the binding of the endogenous agonist adenosine to the two pharmacologically relevant A2A AR conformations [+ binding site center of mass, residues D52, S91, and N280 were chosen; for the orthosteric A2A AR binding site, residues N253, F168, H250, and H278 were selected.Supervised molecular dynamics (SuMD) simulations were exploited to sample and characterize the binding pathway of the Narmations ,22,23,31In all SuMD productive replicates in which adenosine reached the orthosteric binding site, the final state evolution and stability was evaluated through the collection of a 100 ns long classical MD.2A AR, and wrapped into an image of the system simulated under periodic boundary condition. In those cases where a reference was present, the RMSD of the ion or adenosine molecule was computed with respect to the experimental crystallographic complex (4EIY for sodium and 2YDO for adenosine). The RMSD values were plotted over time and reported in the movies present in the All the SuMD trajectories collected were analysed by an in-house tool written in tcl and python languages, as described in the original publication . Briefly+ atomic coordinates, during all replicates collected [2A AR conformations. POVME 2 python software was exploited to perform the calculation, after defining a spherical inclusion region cantered on agonist centroid coordinates and characterized by a 9 Å radius dimension [SuMD trajectories investigating the recognition pathway of sodium were furthermore geometrically analysed to identify significant populations of ion position, among the multitude of sampled data. Prody, a python framework for MD manipulation and analysis, was exploited to compute the pairwise root mean square deviations (RMSDs) of Naollected . From eaollected ,31. The imension .2A AR agonists is not fully understood. X-ray structural studies have pointed out the presence of a binding site for the cation in the core of the TMD of the resting receptor (and many other class A GPCRs). However, the high degree of similarity with the intermediate-active (agonist-bound) state of the receptor , in the attempt to retrieve mechanistic insight.The molecular mechanism that triggers the negative allosteric modulation of the sodium ion on the Areceptor  does not+, whose concentration in the extracellular environment is close to 140 mM, has a fundamental role in controlling the conformational landscape of the A2A AR, characterized by few, highly populated, stable states. The most accepted model describes the sodium as capable of selectively binding only to the inactive-like receptor population. The macroscopic effect of this is a shift of the equilibrium towards the resting state of the receptor, and a decrease in affinity towards agonists. In keeping with this conformational selectivity as well with previous work, our simulations outlined the A2A AR inactive structure as able to coordinate the sodium ion without any topological modification of the putative allosteric site.10,16 On the other hand, during the simulated binding on the intermediate-active conformation, an increase of the inter-TM distances was necessary to accommodate the cation, possibly anticipating a receptor transition toward the inactive-state. The “toggle switch” W2466.48 was pointed out as a possible gatekeeper of the sodium binding event. Interestingly, SuMD suggested different binding paths on the two A2A AR states. It is intriguing to speculate that the inactive state of the receptor could selectively drive the binding of the sodium ion by putatively shaping the charge distribution of the meta-stable binding sites along the path.The Na2A AR . Indeed, seven simulations out of ten resulted in an orthosteric complex, while only one SuMD replicate on the inactive structure was productive. We propose the different flexibilities of the extracellular side of the receptor (where the first interactions able to influence the agonists binding occur) as a driving force of this divergence. The presence of the sodium ion in its allosteric site possibly prevented the receptor from adapting to the incoming agonist, due to an opening up of the EC vestibule and, in turn, of the orthosteric site. As a partial confirmation of this, the TM1, TM2, and ECL2 formed less extensive contacts with the adenosine in the inactive A2A AR  due to t2A AR .The speculative mechanism proposed in this work should be further investigated on other GPCRs.


Antagonistic receptive field surrounds are a near-universal property of early sensory processing. A key assumption in many models for retinal ganglion cell encoding is that receptive field surrounds are added only to the fully formed center signal. But anatomical and functional observations indicate that surrounds are added before the summation of signals across receptive field subunits that creates the center. Here, we show that this receptive field architecture has an important consequence for spatial contrast encoding in the macaque monkey retina: the surround can control sensitivity to fine spatial structure by changing the way the center integrates visual information over space. The impact of the surround is particularly prominent when center and surround signals are correlated, as they are in natural stimuli. This effect of the surround differs substantially from classic center-surround models and raises the possibility that the surround plays unappreciated roles in shaping ganglion cell sensitivity to natural inputs. The receptive field (RF) surround is a ubiquitous feature of early sensory computation. Surrounds in the retina have been proposed to enhance sensitivity to edges in a visual scene via lateral inhibition , and to Here, we answer several questions about center-surround RF structure and its relation to natural scenes. First, how does the circuit location of surround suppression  impact RGC computation? Second, does surround activation change the linearity of spatial integration by RGCs? And finally, how do the statistics of natural scenes influence nonlinear interactions between the center and the surround?Classical Difference-of-Gaussians  and modeFor many RGC types bipolar cells act as excitatory, nonlinear subunits within the RF center  Figure . The degWe hypothesized that surround modulation could dynamically regulate nonlinear spatial integration in a RGC by controlling the effective rectification of the bipolar synapse. Thus, in response to weak surround input or surround input that hyperpolarizes bipolar cells, bipolar cell terminals presynaptic to the RGC are in a rectified state and the RF center integrates nonlinearly over visual space . When suin vitro macaque retinal preparation in conjunction with computational modeling of the RGC RF to explore the impact of the surround on nonlinear RF structure and natural scene encoding. We started by testing the hypothesis outlined in We used single-cell patch electrophysiology in an To test the hypothesis of We previously found that nonlinear spatial integration endows Off parasol RGCs with sensitivity to spatial contrast in natural images . To testAs shown previously, Off parasol cells responded much more strongly to natural images than to linear equivalent stimuli, especially when the natural image contains high spatial contrast . Dark suWe used the difference in spike count between a natural image and its linear equivalent disc as a metric of spatial contrast sensitivity. This difference, as shown in Does the impact of the surround on spatial integration in the center depend on specific statistics of natural images or is it a more general phenomenon? To answer this question, we repeated these experiments using a flashed split-field grating rather than natural image patches . BecauseWe repeated this experiment for surround contrasts ranging from +0.9 to −0.9 . While tTo test whether the effect of the surround on spatial integration was present in the bipolar synaptic output, we repeated these experiments while measuring a ganglion cell’s excitatory synaptic inputs , which iThe experiments described in The hypothesis in We constructed three models of how center and surround inputs combine to determine the cell’s excitatory conductance response when both RF regions are stimulated . (1) In the ‘independent’ model , inputs We fit the nonlinear functions in each model using a subset of simultaneous center-surround trials and used the remaining simultaneous trials to test how well each model could predict the cell’s response. These models generally captured RGCs see , this diRGCs see . The shaRGCs see , despiteRGCs see . Hence tThe hypothesis in These joint response distributions show that the relationship between center activation and excitatory conductance depends on surround activation. When the surround is only weakly activated  to 0 (uncorrelated) to +1 . When noise stimuli in the center and surround were negatively correlated, inputs to the center and surround summed linearly or very nearly so , left. AThese results indicate that nonlinear center-surround interactions depend strongly on center-surround intensity correlations, and hence that the importance of these interactions could be underestimated from stimuli such as spatial noise that lack intensity correlations.The experiments described above show that surround signals, rather than only interacting with the fully formed center signal, can alter how the center integrates over space. Two aspects of these results deserve emphasis: (1) nonlinear spatial integration is maximized when center and surround experience similar mean luminances  and 3 anNatural visual stimuli, such as the change in input encountered after a saccade, typically include changes in mean luminance and spatial contrast. Such stimuli will activate both linear and nonlinear response components, and these may not interact in a straightforward manner. To make this more concrete, consider a population of Off bipolar cells acting as subunits in the center of the RGC RF. At rest, the synapse of each bipolar cell is in a sharply rectified state. A decrease in mean luminance over the RF center will depolarize the synapse associated with each RF subunit, shifting each into a locally linear state , top, anWe tested this prediction in Off parasol RGCs, using modified grating stimuli with nonzero mean luminance. For each grating stimulus, we also presented a corresponding linear equivalent disc stimulus, which has the same mean luminance as the grating, but lacks any spatial contrast. The degree to which a cell’s response to these two stimuli differs is a measure of the sensitivity of the cell to spatial contrast, or, equivalently, the strength of nonlinear spatial integration.We presented these stimuli to Off parasol RGCs while measuring spike responses. A grating with a dark mean luminance signal produced a similar response as a linear equivalent stimulus , left, iTo quantify the spatial contrast sensitivity in these experiments, we used a nonlinearity index  to the RF center while pairing each with three distinct surround conditions: no surround stimulation ; the nateriments , a darkeeriments .These observations are consistent with the idea that natural images provide inputs to the surround that can preserve the spatial contrast sensitivity of the RF center compared to center inputs alone. Key to this relative invariance of contrast sensitivity is the ability of the surround to control the degree of rectification of the bipolar subunits that comprise the RF center and the strong positive correlations between center and surround inputs created by natural images.The appropriate surround activation can preserve spatial contrast sensitivity in the context of both natural image and grating stimuli. Note, however, that the NLI is, on average, lower for randomly selected images than for grating stimuli compare . This isTo explore the relationship between naturalistic surround activation and spatial contrast sensitivity in a manner not possible in our experiments, we constructed a simple spatial RF model composed of nonlinear, center-surround subunits  and MateHere, we have shown that visual inputs to the RF surround can change how the RF center integrates inputs across space, including in natural scenes. During natural vision, the local luminance experienced by a RGC can vary dramatically from fixation to fixation . BecausePast work in parasol RGCs as well as other RGC types has shown that the inclusion of nonlinear subunits in the RF center is important to capture responses to spatially structured stimuli , includiFor the sake of concreteness, and based on past work in mouse retina , we haveImportantly, we chose to focus on how systematically changing the mean intensity in the surround impacts coding in the RF center. This neglects the impact of spatial structure in the surround. Past work shows that RGC RF surrounds can show nonlinear spatial integration when probed with gratings and similar stimuli . If nonlThe LN cascade modeling analysis in We focused here on Off parasol RGCs because of the strong nonlinear spatial integration seen in these cells, and its impact on the encoding of natural scenes . We alsoSimilar RF subregion interactions are present in other visual neurons. For example, activation of suppressive subunits in V1 RFs can change the shape of the nonlinear relationship between excitatory subunit activation and spike response . V1 surrThe center-surround interactions observed here hint at a more general form of response modulation via parallel processing. Indeed, the circuit mechanisms that give rise to the observed center-surround interactions and modulation of spatial contrast sensitivity, namely nonlinear synaptic transfer functions and convergence of parallel neural pathways, are ubiquitous in neural circuits. The key circuit feature that creates unexpected interactions between parallel circuits is convergence prior to important nonlinear circuit elements—for example the bipolar output synapse. Such early convergence can cause one parallel circuit to regulate the effective nonlinearity applied to signals in the other circuit, and in doing so change the nature of the computation implemented by the circuit. Convergence could occur through rapid presynaptic inhibition, gap junctions, or integration of excitatory and inhibitory dendritic inputs in the presynaptic cell.This work supports the broader notion that complex neural computations need not rely on exotic circuitry. Rather, interactions of known circuit elements can produce a variety of computations under the right stimulus conditions by producing modest changes in signaling that enhance or mitigate the impact of nonlinear mechanisms such as the bipolar output synapse. This highlights the importance of exploring how known neural circuit mechanisms interact under diverse stimulus conditions, including during naturalistic stimulation.M. nemestrina, M. mulatta, or M. fascicularis) via the tissue distribution program at the Washington National Primate Research Center. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington. Dissection procedures have been described previously . Spike responses were measured using extracellular or loose-patch recordings with an Ames-filled pipette. For voltage clamp recordings, we used low-resistance pipettes  and protocols used in this study can be found at https://github.com/Rieke-Lab/turner-package (https://github.com/elifesciences-publications/turner-package).Stimuli were presented and data acquired using custom written stimulation and acquisition software packages Stage (stage-vss.github.io) and Symphony (symphony-das.github.io). Lab-wide acquisition packages can be found at Visual stimuli were presented with 60 Hz frame rates on an OLED microdisplay monitor  focused onto the photoreceptors. Monitor outputs were linearized by gamma correction. Stimuli were calibrated using monitor power outputs, the spectral content of the monitor, macaque photoreceptor spectral sensitivity , and a cor cones  and 1 μmBefore every parasol RGC recording, we found the center of the cell’s RF using a split-field contrast reversing grating stimulus at 4 Hz and 90% contrast. To do this, we translated the grating until the two F2 response cycles were balanced (i.e. we minimized the F1 while maximizing the F2 component of the response). We performed this search in both the horizontal and vertical dimensions. In experiments, where we specifically targeted visual stimuli to the RF center or surround, we measured each cell’s area-summation curve and fit, online, to these data a circular difference-of-Gaussians RF model in radial coordinates. This model is described in e.g. see . These bhttp://live.ece.utexas.edu/research/doves/. The images in the DOVES database were selected from the van Hateren natural image database , and more general analysis code used in this study can be found in https://github.com/mhturner/MHT-analysis . Throughout the study, reported p-values were computed using a Wilcoxon signed-rank test.Data analysis was performed using custom written scripts in MATLAB (Mathworks). The code used to analyze the data in this study, perform the computational modeling, and generate the figures presented can be found at The center-surround models in normcdf in MATLAB).For each of the nonlinearities in the models, we parameterized a smooth curve based on a cumulative Gaussian function to fit to the mean response as a function of generator signal (see ). Here, Independent model is described by seven free parameters. Three each for each of the independent nonlinearities of the form described in (1) The Shared model is described by five free parameters. Three for the single nonlinearity ((2) The inearity , a vertiStacked model is described by ten free parameters. Three for each of the three nonlinearities ((3) The earities  and a siTo fit and test these models, we collected For the image computable model in The models in For the simpler spatial RF model  in The RF model is described by four fixed parameters: the size of the Gaussian subunit center, (The rectification index (RI) used in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Andrew King as the Senior and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Receptive field center-surround interactions mediate context-dependent spatial contrast encoding in the retina" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The antagonistic receptive fields of many retinal ganglion cells consist of two components, a center and a surround responding oppositely to light. Models of these receptive fields assume that the surround is added after the center response is established. However, evidence from many species, including mouse and primate retina, suggests that center-surround antagonism is established at the level of cones and bipolar cells through lateral inhibition from horizontal and amacrine cells, i.e. before retinal ganglion cells summate their input. The authors explored the functional consequences of this receptive field organization in OFF parasol retinal ganglion cells of the primate retina with patch-clamp recordings and modeling. Using sophisticated stimulus paradigms, they found that nonlinear spatial integration was maximal when center and surround were stimulated with a similar mean luminance – a correlation often present in natural scenes. These results show that activation of the surround switches the visual computation performed by the receptive field center from encoding information about local luminance to encoding information about spatial contrast in the visual scene.The reviewers agreed that these experiments have been expertly performed, analyzed and presented. The findings are exciting and advance our understanding of how receptive field organization may enable retinal ganglion cells to maintain their contrast sensitivity independently of mean luminance. This paper may change the way we think about the center-surround organization of retinal ganglion cells and has implications for many other circuits in the CNS.Essential revisions:A question was raised about potential relevance to other circuits: The analysis seems to suggest that the presynaptic inhibition exhibits fast temporal dynamics that are required for the computations. This is certainly plausible in the retina, where bipolar cells receive presynaptic inhibition mediated by fast GABA-A and glycine receptors. In many brain circuits, however, presynaptic inhibition is mediated by slower metabotropic receptors. Is this kind of processing less likely to occur in those regions? Information can be processed on multiple time scales, of course, but this raises the question as to whether the presence of presynaptic ionotropic inhibitory receptors is a prerequisite for this kind of signal processing on a faster time scale. It would be useful to address this at the end of the Discussion.Work from Tim Gollisch's lab has shown that surround integration varies with spatial scale. It would have been interesting to probe receptive fields with patches of natural scenes in which light intensity is maintained and the spatial scale of the surround varied (instead of shuffling surrounds and thereby changing light intensity/contrast). The authors should relate their findings to this previous work.One of the reviewers felt that you should state at the beginning of the article that this study focuses on OFF parasol retinal ganglion cells. It was also suggested that you should mention OFF retinal ganglion cells in the title and perhaps transfer data on ON parasol retinal ganglion cells to the supplementary information. However, it was agreed to leave this up to you. Essential revisions:A question was raised about potential relevance to other circuits: The analysis seems to suggest that the presynaptic inhibition exhibits fast temporal dynamics that are required for the computations. This is certainly plausible in the retina, where bipolar cells receive presynaptic inhibition mediated by fast GABA-A and glycine receptors. In many brain circuits, however, presynaptic inhibition is mediated by slower metabotropic receptors. Is this kind of processing less likely to occur in those regions? Information can be processed on multiple time scales, of course, but this raises the question as to whether the presence of presynaptic ionotropic inhibitory receptors is a prerequisite for this kind of signal processing on a faster time scale. It would be useful to address this at the end of the Discussion.This is an interesting point – thank you for bringing it up. We have added a paragraph to the Discussion  that discusses this point. We agree that metabotropic presynaptic inhibition would be better suited for slow-timescale processing than for the dynamic changes in computation we observed in the parasol circuit. Presynaptic inhibition is also not a requisite feature for the form of center-surround interactions that we observe. For example, inhibitory synaptic input to the dendrites of a cell comprising a RF subunit could control the voltage and nonlinearity of the subunit output in much the same way as axonal (presynaptic) inhibitory input to the same cell. The key point is that the combination of signals occurs prior to the subunit nonlinearity. We now note this possibility in the Discussion as well.Work from Tim Gollisch's lab has shown that surround integration varies with spatial scale. It would have been interesting to probe receptive fields with patches of natural scenes in which light intensity is maintained and the spatial scale of the surround varied (instead of shuffling surrounds and thereby changing light intensity/contrast). The authors should relate their findings to this previous work.Thank you for pointing this out. We agree that this ) is an interesting and relevant finding. We added a paragraph to the Discussion (third paragraph in the subsection entitled “Consequences for receptive field models”) that relates our findings to this work. Studying natural images involves many decisions about which features to manipulate; we hope in future studies to build on the work here to more completely explore the full set of features of natural images that control surround activity; nonlinear spatial integration and sensitivity to spatial structure in the surround will certainly need to be included in a more complete understanding.One of the reviewers felt that you should state at the beginning of the article that this study focuses on OFF parasol retinal ganglion cells. It was also suggested that you should mention OFF retinal ganglion cells in the title and perhaps transfer data on ON parasol retinal ganglion cells to the supplementary information. However, it was agreed to leave this up to you.Thank you. We agree that the Introduction of the original manuscript suggested this study would focus on both On and Off parasol RGCs in equal measure, while the results did not follow through with that. However, we prefer to keep the On parasol RGC results in the main text, alongside the Off parasol data, for two reasons:1) We feel that the presence of similar center-surround interactions in the On parasol excitatory inputs is interesting and suggests that these interactions might be a general feature of RGC RFs rather than a peculiarity of Off parasols. The impact on spike output is more complicated for On parasol RGCs because, in part, of strong inhibitory inputs that shape nonlinear responses in On parasols. This issue is beyond the scope of the current study, and we are currently finishing a study dealing with the impact of inhibitory inputs on spatial integration by On parasol RGCs. We have revised the text to point out these issues (see below).2) For presentation purposes, it is much cleaner and simpler to present the On parasol data alongside the Off data in Figures 4-7, since the experiments and analyses are the same. This also facilitates interesting comparisons between On and Off cells, e.g. stronger rectification in Off compared to On parasol excitatory input , greater correlation between excitatory input and spike output in Off compared to On parasol RGCs , and the stronger nonlinear center-surround interactions in Off compared to On parasol RGCs .That being said, we did not communicate the importance or relevance of the On parasol results clearly in the original version of the manuscript. We have revised the text in a few places to more accurately set reader expectations and communicate how the On parasol results are relevant. In particular, see:1) The introduction, where we explicitly say that we focus on Off parasol RGCs.2) The Results introducing Figure 2—figure supplement 1, where we now point out that the presence of similar center-surround interactions in On parasol excitatory inputs suggests a generality to this phenomenon, or at least that it is not a feature unique to Off parasols.3) The Results introducing Figure 4 .4) The final paragraph of the subsection entitled “RF center and surround interact nonlinearly during naturalistic visual stimulation.”5) The Discussion, first paragraph of subsection entitled “Relevance to other neural circuits.”If the reviewers continue to feel that the On parasol data are distracting in the revised manuscript, we will move it to the supplement. We have also kept the title general, but are happy to reconsider if the reviewers feel that the title should indicate a focus on Off parasol cells.


Four-point probing and ultraviolet-visible (UV-Vis) spectroscopy demonstrate that the conductivity and transmittance can be controlled by varying the H2/Ar flow rate, treatment time, and radio-frequency (RF) power. Optical emission spectroscopy reveals that the Hα intensity depends on these processing parameters, which influence the removal of oxidative functional groups (confirmed via X-ray photoelectron spectroscopy) to yield reduced GO (rGO). To further improve the conductivity while maintaining high transmittance, we introduce silver nanowires (AgNWs) between rGO and a PET substrate to obtain a hybrid rGO/AgNWs/PET with a sheet resistance of ~100 Ω/sq and 81% transmittance. In addition, the hybrid rGO/AgNWs thin film also shows high flexibility and durability and is suitable for flexible and wearable electronics applications.We employ H Polyethylene terephthalate (PET) is a thermoplastic polyester resin and widely used in food and packaging industries ,2. RecenAs a result of its excellent optical transparency, flexibility and electrical conductivity, graphene has attracted research attention related to the development of transparent electrodes on flexible substrates. Such devices can be employed in various applications, including solar cells, touch panels, organic light emitting diodes, and wearable electronics ,4,5,6. M2 environment has been demonstrated to be an effective reduction method [Graphene oxide (GO), a derivative of graphene, is a promising alternative for the mass production of graphene-based materials because it can be synthesized in large quantities from inexpensive graphite and is soluble in various solvents. The preparation of a dispersed form of graphene for use in flexible electronic devices is attractive because this material is low cost and solution-processable. However, GO is almost insulating, due to the presence of oxidative functional groups. Therefore, the removal of these groups is necessary in order to convert GO into reduced graphene oxide (rGO). Typically, a chemical method with various reducing agents such as hydrazine, sodium borohydride, and hydroiodic acid, along with vitamin C, glucose, and alkali, is used to reduce GO ,13,14,15n method ,20, the α) enables effective removal of the oxidative functional groups. However, it has been reported that both the ions and vacuum ultraviolet (VUV) radiation in plasma can break the graphene lattice and destroy the honeycomb-like carbon (C) nanostructure with ease, because their energies are higher than the C–C bond energies of sp2-hybridized C atoms in graphene [Compared with the methods previously reported in the literature, plasma is environmentally friendly and has a short reducing time as part of a low-temperature process with a high-purity environment. For GO reduction, the generation of atomic hydrogen  method for the functionalization of nanocrystalline and thin-film materials for applications in flash memory devices and ion sensors, respectively ,28. Both2/Ar mixture gas is introduced into the LDPT apparatus, because the hydrogen radicals can be enhanced by Ar via penning ionization [2/Ar ratio, treatment time, and plasma power. It is reported that silver nanowires (AgNWs) networks, which can be also prepared by low-cost solution-based process compatible with rGO preparation, exhibit high conductivity, high optical transmittance, and good mechanical flexibility [In this study, an Hnization . We systxibility ,34. Howexibility ,36. In tGO solution was purchased from Graphenea Inc., San Sebastián, Spain. The GO was dispersed in deionized (DI) water with a 4-mg/mL concentration. The GO was diluted using isopropanol (IPA) and then coated on an optical grade PET substrate  via spin-coating with 1000-rpm spin speed for 30 s to form GO sheets. Hereafter, these prepared samples are labeled “GO/PET.” Subsequently, the GO/PET was baked to remove the solvent thoroughly using a hot plate at 60 °C for 10 min. The thickness of as-prepared GO film was about 30 nm measured by atomic force microscope. The details were described in 2/Ar-LDPT. The plasma of LDPT was generated by an inductively coupled plasma system , which was conducted through the vacuum-chamber view port. Note that OES is a powerful instrument for characterization of the excited states in a plasma environment.The reduction process to form rGO/PETs was performed using Hstem see . The GO/stem see . Both pl2/Ar-LDPT, the sheet resistance and transmittance were investigated using a four-point probe and ultraviolet-visible (UV-Vis) spectroscopy . The transmittance of the PET substrate used in this study was 90%. The chemical compositions of the rGO sheets were examined using X-ray photoelectron spectroscopy  with a monochromated Al Kα source. Raman spectra were collected using a Horiba Raman system  with a laser wavelength of 532 nm to investigate the structure of rGO sheets. The Si peak at 520 cm−1 was used as the reference for wavenumber calibration prior to each measurement. A field emission scanning electron microscope  and atomic force microscope  were used to study the surface morphologies of AgNW and hybrid thin film.After the rGO sheet preparation using H2/Ar plasma spectra obtained for various H2/Ar ratios using OES. For all spectra, dense lines corresponding to Ar can be observed in the 700–800 nm range. A peak is positioned at 656.3 nm for all H2/Ar mixing ratios, which is due to the presence of Hα. From α intensity increases; this change results from penning ionization [2/Ar ratio of 15/15, the Hα intensity is comparable to that of pure H2 plasma, indicating reduced penning ionization. Accordingly, in what follows, we neglect the pure H2 plasma conditions affecting the rGO characteristics. 2/Ar plasma spectra obtained for various RF powers and a fixed H2/Ar ratio of 10/20. The relative Hα intensity was found to increase with increased RF power as shown in 2/Ar ratio and RF power greatly influence the Hα intensity, which is the main species that reduces GO.nization . When thα intensity and treatment time. The as-coated GO sheet exhibited no conductivity and transmittance of 88.9% at wavelength of 550 nm. 2/Ar ratios at an RF power of 200 W. For all treatment times, the H2/Ar ratio of 5/25 exhibits the lowest sheet resistance. As the Ar flow rate resistance decreases. In particular, in the case of the five-minute treatment time, all the sheet resistances obtained for H2/Ar ratios of 10/20 and 15/15 exceeded the measurement limitation (104 kΩ/sq), as denoted by the “x” symbols in α intensity, which prevented achievement of GO reduction within a short treatment time. In the case of 10-min treatment, the sheet resistance decreases slightly with increased Ar flow rate. This result also corresponds to decreased Hα intensity, as shown in 2/Ar ratio. This result also suggests that the maximum reduction can be achieved for a sufficiently long treatment, even though the plasma exhibits the lowest Hα intensity under 200-W RF power. It is worth noting that the temperature did not increase during the 30-min treatment, because LDPT is a very low-damage treatment that efficiently eliminates the ion bombardment that induces a temperature increase. 2/Ar ratio at an RF power of 300 W. The result has almost the same trend as that observed in 2/Ar ratio of 10/20 and five-minute treatment. The sheet resistance of the rGO/PET sample under these conditions was measurable. For the 30-min case, the sheet resistance values also remained at the same level (ca. 50 kΩ/sq) as those obtained for 200-W RF power, for all H2/Ar ratios. 2/Ar ratios under 400-W RF power. For the five-minute case, the sheet resistance values decreased slightly with increasing Ar flow rate, due to the increase of Hα. On the other hand, the sheet resistances for the 10- and 30-min cases are almost at the same level and are not related to the H2/Ar ratio. This result suggests that the reduction time can be shortened significantly when a high RF power is used. The prepared rGO sheets showed good stability by confirming the variation of sheets resistance as the samples were stored in atmosphere  located at 284.5 eV, the XPS C 1s spectra of the GO sheets feature signals at higher chemical shifts for oxidative functional group, including hydroxyl groups (C–OH), carbonyl groups (C=O), and carboxyl groups (COOH) [2/Ar-LDPT process, the intensities of the oxidative functional groups located at the higher chemical shifts decreased, indicating GO reduction. It is also apparent that more oxidative functional groups were removed when the treatment time was longer as shown in 2/Ar ratio and RF power dependency on the removal of the oxidative functional groups. As the Ar flow rate and RF power increased, more oxidative functional groups were removed, because of the higher Hα intensity. These results are consistent with those for the sheet resistance and Hα intensity, shown in The oxygen bonding in the rGO sheets after the Hs (COOH) ,39. Afte2/Ar-LDPT process conditions, structural changes to the GO sheets resulting from H2/Ar-LDPT were investigated by Raman spectroscopy. 2/Ar ratio, and RF powers. The Raman spectrum of as-coated GO sheet shows a G-band (ca. 1600 cm−1) and D-band (ca. 1350 cm−1) corresponding to ordered sp2 bonded carbon and structural defects created by oxygenated functional groups, respectively [2/Ar-LDPT process, as shown in I(D)/I(G)) is commonly used as an index for the defect level existing in graphene. As exhibited in I(D)/I(G) of as-coated GO sheet is 1.13 and the ratio slightly decreased as the treatment time increased. The I(D)/I(G) is 1.01 for treatment time of 30 min. The dependencies of I(D)/I(G) on H2/Ar ratio and RF power are displayed in I(D)/I(G) decreased prominently. The lowest values of I(D)/I(G) for Ar flow rate of 25 sccm and RF power of 400 W are 0.94 and 0.89, respectively. Those results indicated that the more the Hα generates in H2/Ar LDPT process, the slightly lower the defect level of rGO sheet is obtained. Moreover, it has been reported that reduction by hydrazine hydrate or hydroiodic acid leads to higher I(D)/I(G) corresponding to the generation of defects [I(D)/I(G) of GO sheets treated by H2/Ar-LDPT indicates that the reduction process slightly repairs the flakes of GO sheets without introducing additional defect due to plasma damage.Following the characterization of chemical compositions of GO sheets as a function of various Hectively . After t defects . The dec2/Ar-LDPT process. The process conditions used to obtain hybrid rGO/AgNWs thin films on the PET were as follows: RF power: 300 W; H2/Ar ratio: 10/20; treatment time: five minutes. The AgNWs were diluted in IPA in three ratios (AgNWs:IPA): 1:10, 1:15, and 1:20. 2/Ar-LDPT, the sheet resistance values of the resultant rGO/AgNWs/PET samples were almost identical. This result indicates that the AgNWs were embedded into the rGO during spin-coating, which compensated for the discontinuity between each AgNW and yielded considerable enhancement in the conductivity. The transmittance spectra were measured by UV-Vis spectrophotometer  of the hybrid thin film was slightly decreased after H2/Ar-LDPT process.To investigate the influence of HRc). The Rc was estimated by measuring the outer diameter of the objects. In this measurement, the samples were bent to wrap various cylindrical objects, including battery, pen and laser-pointer, and then relaxed for bending cycles of 1, 5, 10 and 100. After each bending cycle, the sheet resistance of rGO/AgNWs/PET was measured. Rcs of cylindrical objects expressed by Rs/Rs0, where Rs and Rs0 are sheet resistances of before and after bending, respectively. The insets show the photos of rGO/AgNWs/PET wrapping on cylindrical objects with various Rcs. It is clearly seen that the Rs/Rs0 shows no apparent dependence on bending cycle or Rc, and all the values only slightly increases above 1. The results indicate that the morphology of the network of rGOs and AgNWs in hybrid rGO/AgNWs thin film was maintained during the bending test, demonstrating high flexibility and durability.Finally, the mechanical flexibilities of rGO/AgNW/PET samples were studied by placing them on cylindrical objects with various radii of curvature (


Research on the aryl hydrocarbon receptor (AhR) has largely focused on its activation by various environmental toxins. Consequently, only limited inferences have been made regarding its constitutive activity in the absence of an exogenous ligands. Evidence has shown that AhR is constitutively active in advanced prostate cancer cell lines which model castration resistant prostate cancer (CRPC). CRPC cells can thrive in an androgen depleted environment. However, AR signaling still plays a major role. Although several mechanisms have been suggested for the sustained AR signaling, much is still unknown. Recent studies suggest that crosstalk between constitutive AhR and Src kinase may sustained AR signaling in CRPC. AhR forms a protein complex with Src and plays a role in regulating Src activity. Several groups have reported that tyrosine phosphorylation of AR protein by Src leads to AR activation, thereby promoting the development of CRPC. This review evaluates reports that implicate constitutive AhR as a key regulator of AR signaling in CRPC by utilizing Src as a signaling intermediate. Androgens and androgen receptor (AR) signaling play a predominant role in male sexual development, growth of the prostate gland and progression of prostate cancer to CRPC2. In CRPC, AR signaling has been shown to be sustained by a variety of mechanisms including increased androgen uptake by prostate cancer cells, increased AR expression, AR gene mutation and activation by other transcription factors4. The aryl hydrocarbon receptor is a transcription factor that has been extensively studied for its role in mediating the toxic effects of a wide range of environmental toxins. However, recent evidence has shown that AhR possess intrinsic functions independent of activation by an exogenous ligand. Constitutively active AhR has been shown to interact with hormone receptors and may play a role in progression of hormone-related cancers. Particularly, constitutively active AhR interacts with AR and act as a functional transcription unit5, a mechanism that may lead to enhanced AR signaling in CRPC.Most men who die of prostate cancer present with castration resistant prostate cancer (CRPC)6. As an inactive complex, AhR is found in the cytosol where it interacts with tyrosine kinase c-Src as well as two molecules of HSP90, co-chaperone p23 and immunophilin-like AhR interacting protein (AIP/XAP2)8. In its active form, AhR disassociates from its chaperone proteins and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT). The heterodimer activates gene expression through interactions with related xenobiotic responsive elements (XREs) located on Ah- responsive gene promoters9. Studies concerned with the intrinsic functions of AhR have found that overexpression of the receptor may promote carcinogenesis in the absence of a exogenous ligand. AhR protein and mRNA expression is associated with phases of rapid proliferation and differentiation in certain tissues. Conversely, AhR-defective cell lines demonstrate a reduced proliferation rate10. Ectopic over expression of AhR in immortalized normal mammary epithelial cells induced a malignant phenotype with increased growth and acquired invasive capabilities11. A separate study using a constitutively active AhR construct lacking a ligand binding domain revealed that AhR acts as a transcriptional coregulator for the unliganded AR. These studies show that the endogenous AR along with the constitutively active AhR were recruited to androgen-responsive elements to initiate signaling in an androgen depleted environment5.Although AhR has been extensively researched for its role as a xenobiotic receptor; RNA interference, overexpression, and inhibition studies suggest a role for AhR in multiple tumor types beyond activation by environmental contaminants9 in both constitutive and ligand activated signaling. AhR ligands such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), components of cigarette smoke such as benzo(a)pyrene (BaP), and a wide range of polycyclic aromatic hydrocarbons (PAH) exert their biological influences by binding directly to the AhR. Following ligand activation, AhR’s primary role is identified as the control of xenobiotic metabolism through cytochrome P450s, some of which are transcriptional targets of AhR13. Activation of AhR by PAHs has been reported to antagonize AR signaling. For example, TCDD has been shown to alter sex steroid hormone secretions. TCDD was also shown to block the androgen dependent proliferation of prostate cancer cells14. Simultaneous activation of AhR and AR with TCDD and an androgen derivative, respectively, decreased AR protein levels15. Thus, activation of AhR by a ligand results in decreased protein expression of both AhR and AR. This action may explain the anti- androgenic actions of a number of PAHs, is distinctive from the effects seen with constitutive AhR signaling that accompanies overexpression of the AhR protein and may be the result of enhanced activity of AhR chaperone protein, Src kinase.The aryl hydrocarbon locus includes AhR, ARNT, and the AhR repressor (AHRR), which are critical for regulation of AhR signaling16. The PAS domains facilitate interactions with other PAS domain proteins, such as the AhR binding partner, ARNT PAS-A is primarily responsible for protein-protein interaction and PAS-B also encompasses the ligand binding17. cSrc may interact directly with the AhR transactivation and PAS domains7. Western blot and FRET analysis confirm time- dependent phosphorylation of Src following activation of AhR which was blocked in the presence of a specific AhR antagonist18. Furthermore, coimmunopercipitation experiments revealed that AhR regulates Src activity by phosphorylating Src (Tyr 416) and dephosphorylating Src (Tyr527)19.The mammalian AhR protein contains four major structural motifs important in AhR’s interactions with other proteins and transcription factors. The N-terminal basic-helix-loop-helix is the site of DNA binding domain and also participates in dimerization and HSP90 binding. The transactivation domain spans from amino acid 490 to 805 and includes a central glutamine rich region. The two Per-Arnt-Sim (PAS) domains are named after their homology with the clock protein period (Per), the xenobiotic and oxygen sensing ARNT, and the neuronal cell lineage regulator single-minded (Sim) 16. The P20. There are also many reports that SFKs are abnormally activated in prostate cancer cells. SFKs are activated in response to numerous stimuli including neuroendocrine ligands, reactive oxygen species, cytokines and growth factors. These molecules have proven roles in cancer progression, including cell proliferation, adhesion, migration, and invasion21.The increased expression of Src and other Src family kinases (SFK) in a number of prostate cancer cell lines has suggested a role for Src in prostate cancer initiation and progression8. TCDD activation of AhR results in time- and dose-dependent phosphorylation of Src (Tyr416). Reported Src-mediated crosstalk between AhR and EGFR signaling pathways demonstrated an important link between AhR canonical function and TCDD-mediated tumor promotion23. Through direct physical interaction with AR, Src is able to phosphorylate AR and thereby induce ligand-independent activation of AR, a key mechanisms of CRPC24. Conversely, AR overexpression also plays a role in the oncogenic potential of wild-type Src. This suggests that crosstalk and activation of AR and Src is reciprocal25.Src and AhR coexist in a protein complex that also contains HSP90, AhR-interacting protein, and p23 that aides to maintain an inactive AhR complex26. As a phosphoprotein, AR has several serine/ threonine and tyrosine residues that are phosphorylated. Most of the phosphorylated residues are located in N-terminal domain that regulates AR cellular localization, stability and its transcriptional activity.AR contains at least sixteen phosphorylated residues. Several of the residues are phosphorylated after treatment of cells with androgen, antiandrogen, or reagents which activate other signaling pathways and alter transcriptional activity, cellular localization, and stability of AR28.Recently, several groups have reported that tyrosine phosphorylation of AR protein by non- receptor tyrosine kinases Src may have a role in AR activation in the low androgen environment, thereby promoting the development of CRPC. Src-mediated phosphorylation of AR at Y534 resulted in the activation of AR followed by nuclear translocation and DNA binding in the absence of androgens 26–28.29. In the presence of low levels of androgen, AR is normally localized to the cytosol in a complex with molecular chaperones, Hsp40, 70 and 90 in an inactive form. Upon androgen binding, the androgen induces conformational changes in the protein, forms a homodimer, and translocates to the nucleus30. The nuclear translocation results in binding of AR as a transcription factor to ARE’s in the regulatory region of target genes. AR signaling plays a critical role in prostate cancer cell proliferation, survival, and differentiation31.AR is a member of the steroid hormone receptor family and shares a similar domain organization with other members of the nuclear receptor (NR) which is primarily responsible for mediating the physiological effects of androgens by binding to AREs32.Several studies based on molecular cloning of AR cDNA, suggest that its transcriptional activity is critical for all stages of prostate cancer development and progression. Several neutral next- generation sequencing platforms have been used to pursue genomic characterization of prostate cancer at various stages. Results have consistently confirmed a critical role for AR activity in prostate cancer progression33. Whole genome sequencing of 11 early onset prostate cancers suggested that androgens, through AR, contribute in shaping somatic alterations34. These results are further supported by studies demonstrating that AR is known to stimulate the expression of TMPRSS2: ERG which is a common gene fusion associated with prostate cancer initiation via androgen-driven overexpression of the gene fusion products35. It is clear that AR signaling plays a critical role in the development and progression of prostate cancer and serves as a central component of progression to CRPC.While AR signaling is known to modulate the expression of genes associated with cell cycle regulation, survival and growth, its specific functions are not fully defined in prostate cancer38. Direct heterodimerization of AhR and AR has been shown to occur in cells and may partially explain the crosstalk between the two receptors5. Additionally, interaction can happen via coactivators. AhR and AR share a number of coactivator proteins such as SRC1 and p30039. Another possible mechanism AhR may utilize for AR activation is phosphorylation of AR by Src kinase40. Src was shown to mediate crosstalk between AhR and epidermal growth factor receptor in colon cancer cells19. Other studies have shown that Src kinase can promote AR transactivation in C4–2 cells prostate cancer cells. Consequently, inhibition of Src kinase function with a specific inhibitor resulted in decreased AR activation41. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527)42. Immunoprecipitation assays also revealed the association of AR with Src, suggesting complex formation among them43.AhR signaling influences androgen signaling directly at the level of the protein and indirectly through actions on the endocrine system. AhR plays a significant role in sustained AR signaling and growth. However, the mechanism for this role is not clearly understood. One possible mechanism is direct interaction between AhR with AR. AhR has been reported to directly interact with a number of nuclear proteinsin vitro and in vivo studies have established a role for AhR in prostate and prostate cancer development. The development of AhR-null mice has revealed that the function of this receptor is not limited to mediating the effects of PAHs and other ligands44. AhR knockout mice exhibit decreased fertility, decreased liver size, and structural and functional deficits in several tissues45. These include reproductive tract problems such as decreased levels of mature follicles and formation of uric acid stones in the urinary bladder46.The precise molecular mechanism utilized by constitutive AhR signaling to activate AR signaling needs to be investigated further and could include induced activation via protein phosphorylation, direct heterodimerization and interacting via coactivators. Extensive 47. Ligand activation of AhR by TCDD is adequate to disrupt key stages of ductal morphogenesis. Significantly smaller prostate lobes were observed in rats and mice exposed to TCDD during fetalpubertal development. Indicating that TCDD-induced AhR activation delays prostate growth48. TCDD exposure also led to a decreased number of prostate ducts in monkeys49. AhR may serve as a key regulator of AR signaling in CRPC by utilizing Src as a signaling intermediate. Recent studies have revealed that simultaneous inhibition of AhR and Src is sufficient to abolish AR signaling in CRPC cells50. Activated Src is a common signaling intermediate in a number of pathways including AR signaling pathway and has increased activity in CRPC. Although several mechanisms have been suggested for the sustained AR signaling, tyrosine phosphorylation of AR protein by AhR- activated-Src may have a significant role in CRPC that also exhibit constitutive AhR signaling  and in normal, hyperplastic, and cancerous adult human prostate tissueignaling .


Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes—from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.  •Dynamic behavior of heteromeric sHsps hinders structural biology of cytoprotection.•Full-length human HspB2/B3 in 3:1 ratio was crystallized and solved at 3.9-Å resolution.•Assembly is by flexible “nuts and bolts” from terminal regions filling domain pockets.•N-terminal regions bind in an unfolded conformation into shared dimer grooves.•IXI/V motifs from unstructured proteins may be sequestered by sHsps during disease. Mammalian small heat shock proteins (sHsps) are among the first responders to cellular stress and therefore play important roles in human health and disease via the co-chaperone Bag3 sHsps are found in all forms of life and typically are dynamic oligomers that have ATP-independent roles in cellular protein homeostasis, protecting the proteome from uncontrolled aggregation especially during environmental stress The sequences of sHsps from all kingdoms of life contain a conserved, well-structured “α-crystallin domain” (ACD), flanked by a variable, and generally longer N-terminal region and a shorter more conserved C-terminal region Caenorhabditis elegans, shows how 16 AP dimers assemble into a 32-mer by docking C-terminal L-P-I motifs into β4/β8 pockets The crystal structure (PDB ID 4YDZ) of a metazoan sHsp, Sip1 from Although the crystallized sHsp homo-oligomers are symmetrical, most  sHsps are polydisperse. The symmetric sHsps have given indications of flexibility in the system that can contribute to geometric diversity: variation in AP interface register , different temperatures of crystallization , seeding drops with microcrystals, spiking mother liquor components before setting up drops, including the use of additive compounds into the crystallization conditions  and the dehydration of the crystals . The best additives were a combination of gamma-polyglutamic acid and GTP. Attempts to find an entirely new crystallization hit, with a more ordered crystal lattice failed. Throughout this screening process, success was measured by the diffraction range of the crystals. More than 100 crystals were tested at three separate synchrotrons over a period of 2 years. The best crystal diffracted to 3.9 Å and the next best crystal diffracted to 4.1-Å resolution. MS confirmed that the crystals preserved the unusual 3:1 HspB2:HspB3 stoichiometry , exploring different biological buffer systems and pH, varying the ratio of mother liquor/protein solution and changing the total volume of crystallization drop. When these experiments failed to improve crystal resolution, the search was extended to include hiometry b.Solving the structure was challenging due both to the limited resolution of the data and the large size of the asymmetric unit of the crystal comprising three tetramers, giving a total of twelve protein chains. Initially, three chains of one tetramer were identified by molecular replacement with an ensemble of ACDs that comprised Hsp27 (PDB ID 3Q9P), Hsp20 (PDB ID 2WJ5) and HspB5 (PDB ID 2WJ7) overlaid as the search model. The resulting molecular replacement solution contained the AP dimer and one other chain. A bootstrapping method  allowed the identification and modelling of the fourth chain, and finally each of the three tetramers. These are labelled tetramer 1 chains ACDQ, tetramer 2 chains EGFT and tetramer 3 chains IKJV. While the large number of chains was a liability during determination of the asymmetric unit, the 12 chains were then an advantage in refinement, by exploiting the threefold non-crystallographic symmetry in the asymmetric unit throughout refinement and building. The resulting structure was refined using Buster software  to Rfactor/Rfree of 29.7/31.3, an acceptable outcome for the resolution and the fact that not all the model was identified and built.Of the 2088 residues expected in the asymmetric unit, 1330 residues were modelled. Side chains for approximately half of these were able to be modelled. The unmodelled peptide, particularly from the N- and C-terminal regions of HspB2 chains D , is presumably disordered in the crystal, consistent with the resolution being low. Although only 33 residues out of the 85 of the ACDs are identical between HspB2 and HspB3, chains D and Q in tetramer 1  could not be assigned confidently. Therefore, chains D and Q  were modelled as polyAla, along with HspB2/HspB3 conserved prolines. Based on the location in the tetramer, chain Q  is the most likely candidate for HspB3 and is labelled as such, with chain D  assigned HspB2 numbering. Details of the equivalent chains in all three tetramers that have been modelled in the asymmetric unit are in Supplementary Table 1.de novo, into difference electron density that was revealed after refinement using the ACDs only. Some, such as the C-terminal regions of chain A and chain C, have continuous density connecting to the ACDs and a map quality that allowed residue side chains to be modelled. Segments of N-terminal regions were assigned to chains A and C, but the register and direction were uncertain because of the absence of continuous density connecting to the ACD and lack of clear side-chain density in the map. We have modelled as polyAla chain C, residues 20–32 and 36–43 of HspB2, although the numbers and chain direction are uncertain. There are some peptides in the map that could not be assigned to the four chains of each tetramer. These are modelled as chains M,  in the center of tetramers 1 , chains W  at the “top” of tetramers 1  and chains 1   respectively (Supplementary Table 1).Segments of N- and C-terminal regions of HspB2 and HspB3 were built VYISL from the C-terminal region of human HspB2 binds to the β4/β8 pockets of human HspB5 ACD dimer Hetero-tetramer 1 comprising chains ACDQ is now described, with chain Q interpreted as HspB3. Each of the four ACDs is a β-sandwich of seven strands β2–β9, as present in other mammalian sHsps. Two HspB2 chains, labelled A and C a, were iThree register shifts have so far been observed at sHsp AP interfaces IILRHLIEIPVR-15 (IILR7 from HspB3 (chain Q) filling the pockets of chain D  per tetramer that is unresolved and potentially unsatisfied. It is too distal from chain Q to patch its β4/β8 pocket. There is density for residues filling the β4/β8 pocket in chain Q, labelled 1–5 (chain 1) of unknown origin  can suppress aggregation of amyloid peptide β1–40 by interactions involving the β4/β8 pockets The IXI/V motif into pocket interaction is known to be part of the integration of sHsps into the proteostatic cellular machinery. Bag3, which is constitutively expressed in skeletal muscle cells and cardiomyocytes, regulates the cellular pathway that uses the specificity of Hsp70 machinery to recognize and triage misfolded proteins This model of action is consistent with the observation that in the last step in the purification of wild-type human HspB2/B3 hetero-tetramer, a Bag3 column removed all free HspB2, indicating that homo-oligomeric HspB2 has accessible β4/β8 pockets for binding IPV motifs, which are masked when in complex with HspB3. However, in the case of rat HspB2/B3, the complex was less stable as Bag3 also removed HspB2 from the HspB2/B3 hetero-tetramer (data not shown). The greatest difference between the amino acid sequences of rat and human HspB3 is in the N-terminal regions adjacent to the ACD, and in the IPV motif which is TPV in rat Fig. a. These The hetero-tetramer structure suggests that it may function in proteostasis by providing accessible binding sites for partially structured and unstructured polypeptide chains whose origins could be destabilized proteins or  disordered regions of other proteins in quality control networks. Unlike the several vertebrate ACD structures currently available, the HspB2/B3 heteromer comprises full-length proteins. By revealing the heterogeneous nature of the interface sites, this structure provides insight into the plasticity of interactions made by sHsps. They reveal the importance of unstructured and flexible N-and C- terminal regions to heterogeneity and probably point to multi-valency of binding substrates, as in the unrelated molecular chaperone SecB The cDNAs encoding human HspB2 and HspB3 were cloned into the pET3a expression vector using NdeI–BamHI sites and XhoI–EcoRI sites, respectively. The HspB3 sequence coding for the C-terminal lysine residue was replaced by the sequence coding for the last 12 amino acids of HspB2 171-PEEEEEAAIVEP-182 using the reverse primer - CGGAATTCTCAGGGCTCAACTATGGCTGCCTCCTCCTCTTCCTCTGGAGTCCCAACTGGATCCTTTAC. The R7S mutation in HspB3 was introduced by site-directed mutagenesis using the forward primer GGCAAAAATCATTTTGTCTCACCTCATAGAGATTCC and the reverse primer GGAATCTCTATGAGGTGAGACAAAATGATTTTTGCC.Escherichia coli BL21(DE3) strain, transformed with pET3a-HspB2/B3 wild type or mutant, by the addition of IPTG to a final concentration of 350 μM and subsequently incubated for 3 h at 37 °C. Cells were lysed by sonication in TEG buffer  and centrifuged at 16,000g for 45 min at 4 °C. The obtained supernatant was fractionated on a DEAE–Sepharose column, using a gradient of 0–1 M NaCl in 25 mM Bis–Tris (pH 6.35) and 2 mM EDTA and, after desalting, further fractionated on a Source 15Q HR 16/10 column using the same salt gradient. Any excess-free HspB2 in an HspB2/B3 batch was removed by briefly incubating the HspB2/B3 batch at room temperature with recombinant GST-Bag3 coupled to Glutathione agarose beads  in 25 mM Tris (pH 8), 100 mM NaCl and 0.1 mM EDTA, after which the beads were removed. Recombinant GST-Bag3 was obtained with from a Bag3 pGEX4T3 expression construct .Protein expression was induced in the 2O/10% D2O, 20 mM phosphate buffer (pH 7.5) and 100 mM NaCl were acquired at 25 °C and a 1H frequency of 600 MHz on a Varian Unity Inova 600 NMR spectrometer. The two-dimensional 1H–1H TOCSY spectrum had a spin lock mixing time of 80 ms and the 1H–1H nuclear Overhauser effect spectroscopy spectrum had a mixing time of 100 ms.NMR spectra of 4.4 mg/mL of rat HspB2/B3 in 90% HProteins were analyzed on a Q-ToF 2 mass spectrometer (Waters Corporation) modified for transmission of high-molecular-mass species °C: 2 μL of protein 10 mg/mL in 25 mM Tris–HCl (pH 6.9), 0.25 mM tris(2-carboxyethyl)phosphine and 2 mM EDTA was mixed with 2 μL of 0.6 M sodium formate, 0.1 M Tris–HCl (pH 7.8), 7.8% gamma-polygutamic acid (Na + form LM) and 2 mM GTP as additives. The best resolution of many data sets collected at a number of synchrotrons was indexed with XDS, and the cell dimensions of the crystal unit cell were determined to be 177.14, 177.14, and 126.46 Å, with angles of 90°, 90°, and 120°, and the space group is P31. The dataset was solved using Phaser, revealing 12 chains in the asymmetric unit. X-ray data and refinement statistics are given in Crystallization was by hanging drop at 21 The coordinates and structure factors have been deposited to the wwPDB with accession number PDB ID 6F2R.A video of the ACDQ tetramer, colored as depicted in Supplementary VideoSupplementary material- Supplementary Table and FiguresImage 1The following are the supplementary data related to this article.