A priori TIH must result from excessive saliuresis and/or water reabsorption. We hypothesized that pathways regulating the thiazide-sensitive sodium-chloride cotransporter NCC and the water channel aquaporin-2 (AQP2) may be involved. Our aim was to assess whether patients with TIH would show evidence of altered NCC and AQP2 expression in urinary extracellular vesicles (UEVs), and also whether abnormalities of renal sodium reabsorption would be evident using endogenous lithium clearance (ELC).Thiazide diuretics are among the most widely used antihypertensive medications worldwide. Thiazide-induced hyponatremia (TIH) is 1 of their most clinically significant adverse effects. Blood and urine samples were donated by patients admitted to hospital with acute symptomatic TIH, after recovery to normonatremia, and also from normonatremic controls on and off thiazides. Urinary extracellular vesicles were isolated and target proteins evaluated by western blotting and by nanoparticle tracking analysis. Endogenous lithium clearance was assessed by inductively coupled plasma mass spectrometry.2 relative to appropriate control groups; smaller differences in NCC and AQP2 expression persisted after recovery from TIH. These findings were confirmed by nanoparticle tracking analysis. Renal ELC was lower in acute TIH compared to that in controls and convalescent case patients.Analysis of UEVs by western blotting showed that patients with acute TIH displayed reduced total NCC and increased phospho-NCC and AQP2 expression would be expected to result in saliuresis and water reabsorption in TIH patients. This study raises the possibility that UEV analysis may be of diagnostic utility in less clear-cut cases of thiazide-associated hyponatremia, and may help to identify patients at risk for TIH before thiazide initiation.Reduced NCC expression and increased AQP Thiazide diuretics have been used in the management of hypertension for more than half a century.5A priori TIH must result from excessive saliuresis and/or water reabsorption (or ingestion). It therefore seems likely that excessive saliuresis and/or water reabsorption (or ingestion) occurs via altered regulation of NCC and/or aquaporin-2 (AQP2). As thiazide diuretics have not, to the best of our knowledge, been reported to produce saliuresis with a urinary sodium concentration greater than that of plasma, this suggests that excessive water reabsorption (or ingestion) is a fundamental part of the pathogenesis of TIH. Our aim in this study was to probe the molecular mechanisms underlying TIH by investigating whether patients with TIH exhibit evidence of altered regulation of NCC and AQP2.The mechanisms underlying TIH remain unclear. Risk factors for TIH include advanced age, reduced body mass, and concurrent use of other medications that impair water excretion.1 and NCC2 vary by only 1 amino acid.3, however is 9 amino acids longer; it constitutes approximately 40% of NCC is the human nephron. Regulation of NCC occurs principally by 2 mechanisms: regulation of apical membrane abundance (type 1 regulation) and regulation of transporter kinetics (type 2 regulation), the latter being controlled by phosphorylation of key N-terminal serine and threonine residues .2 pathway in the collecting duct.2, increasing the water permeability of the collecting duct.2 (PGE2) is a key modulator of this pathway,2-specific prostaglandin transporter (PGT). A recent study has suggested modest genetic associations with TIH, including aberrant PGE2 reuptake in the distal nephron that may increase AQP2-mediated water reabsorption in a subset of patients who carry a variant within SLCO2A1, the gene encoding PGT.The sodium-chloride cotransporter  is located in the renal cortex; therefore thiazides reduce maximal urinary diluting ability by increasing sodium excretion without affecting the renal medullary sodium gradient required for water reabsorption from the collecting duct.Urinary extracellular vesicles (UEVs) are spherical, structured membrane vesicles that are formed by cells throughout the nephron.22Because lithium is absorbed along with sodium throughout the nephron, it is possible to use measurement of lithium clearance to assess tubular sodium reabsorption. Renal lithium clearance likely reflects mostly proximal tubular sodium reabsorption, because this is where the majority of sodium is reabsorbed.2, we report analysis of urinary extracellular vesicle NCC and AQP2 in TIH patients and controls. We also report assessment of renal tubular sodium reabsorption in TIH patients by measurement of ELC.To fulfill our aim of investigating whether patients with TIH exhibit evidence of altered regulation of NCC and AQPThis study was conducted in line with the standards of ICH/Good Clinical Practise sections 8.2.8 in adherence to the Declaration of Helsinki and was approved by the UK National Research Ethics Committee (reference 11/EM/0233). Informed consent was given by all participants. Between April 2012 and August 2015, a daily search of the biochemistry database of all patients admitted to the department of internal medicine at Nottingham University Hospitals NHS Trust, UK, was undertaken to identify those patients with serum sodium <130 mM (cases). Patients were reviewed by the investigators to establish those whose hyponatremia was due to a thiazide diuretic (TIH). Normonatremic thiazide and nonthiazide controls (serum sodium 135−145 mM) were identified by primary care surgeries in Nottinghamshire and matched as closely as possible to the case patients for age, sex, comorbidities, and polypharmacy. Patients with TIH were also assessed in the outpatient clinic 2 months after thiazide cessation (termed normonatremic TIH case patients off thiazides). The patients described here were a subset of those previously reportedSecond-morning spot urine samples (30 ml) were collected in sterile containers from TIH patients and control groups . Proteasg for 5 minutes at room temperature. Frozen urine samples were thawed on ice, 1 ml dithiothreitol  was added, extensively vortexed and incubated for 30 minutes at 37 °C before UEV isolation. All samples were centrifuged (2500 g for 15 minutes at 4 oC) for urinary cell removal. Cell pellet was suspended in lysis buffer  and stored at −80 oC. Further filtration of supernatant using 0.22-μm filters  was undertaken to remove debris. A 25-ml quantity of filtered supernatant was centrifuged at 15,000 g for 30 minutes at 4 °C. The urine supernatants were then added to Vivaspin (USA) nanomembrane concentrators and centrifuged at 4800 g for 1 hour at 4 °C. Concentrated UEVs were treated with 50 μl dithiothreitol, added directly into the filters, and incubated for 10 minutes at 37 °C. Isolated urinary extracellular vesicles were directly suspended in lysis buffer and stored at −80 °C. Total urine and flow-through fractions were also stored at −80 oC and −20 oC. Total soluble protein concentration was quantified with bicinchoninic acid assay .Urinary nanovesicles, including UEVs, were isolated from both fresh and frozen urine by Ultrafiltration . Before processing of urine samples, nanomembrane concentrators were washed with phosphate-buffered saline solution to remove glycerol and other preservatives and then centrifuged at 2500 2) acrylamide gels. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis was performed on polyvinylidene difluoride membrane . Urinary extracellular vesicle AQP2 was detected by probing the blots with 1:500 diluted anti-AQP2 antibody  followed by anti-rabbit IgG horseradish peroxidase (HRP)−conjugated antibody (Sigma-Aldrich). Urinary extracellular vesicle NCC3 was probed with 1:500 diluted anti-NCC primary antibody (Millipore) followed by anti-rabbit IgG HRP−conjugated antibody (Sigma-Aldrich). Urinary extracellular vesicles NCC1 and NCC2 were probed with 1:500 diluted anti-NCC primary antibody  followed by anti-rabbit IgG HRP-conjugated antibody (Sigma-Aldrich). Urinary extracellular vesicle phospho-NCC  was detected by probing blots with anti–phospho-NCC antibodies , each at 1:500 (0.2–1.2 μg/ml) followed by anti-sheep IgG HRP-conjugated antibody (Sigma-Aldrich). To detect specific phosphorylated forms of NCC, the corresponding nonphosphorylated peptide was incubated with primary antibody (10 μg/ml). Urinary extracellular vesicle PGT was probed with 1:200 diluted anti-PGT primary antibody  followed by anti-rabbit IgG HRP-conjugated antibody (Sigma-Aldrich). The anti-AQP2 and NCC antibodies used target intracellular domains of AQP2 and NCC. Vesicles did not undergo prior permeabilization. Urinary cell pellet was used as a negative control for western blots. Urinary extracellular vesicle ALIX was detected by probing the blots with 1:1000 diluted anti-ALIX antibody  followed by anti-rabbit IgG HRP-conjugated antibody (Sigma-Aldrich). Western blot data were corrected for ALIX as indicated in the legends of P value of <0.05 was considered significant.A total of 25 μg soluble protein per sample was electrophoresed on 7.5 % (NCC and phospho-NCC), 10% (apoptosis-linked gene 2-interacting protein X [ALIX] and PGT) and 12.5%  is a light-scatter microscopy method of tracking microparticles and nanoparticles on the basis of direct and real-time tracking of the particles’ Brownian movement, which results in a description of the particle size and concentration distribution in a given solution. Nanoparticle tracking analysis can be used to count and measure specific subgroups of nanoparticles using fluorescent antibodies against nanoparticle proteins, including extracellular vesicles derived from kidney cells in culture and in urine.2 and NCC-expressing UEVs were analyzed using the NanoSight LM 10 instrument . The analysis settings were optimized and kept constant between samples, and each video was analyzed to give the mean, mode, median, and estimated concentration for each particle size. All experiments were carried out at a 1:1000 dilution of unprocessed urine, yielding particle concentrations in the region of 1.3 × 108 particles/ml in accordance with the manufacturer’s recommendations. For fluorescent NTA analysis, a 532-nm (green) laser diode excited the fluorescent-loaded ECVs with a long-pass filter (430 nm). For quantification of AQP2  and NCC , primary antibodies were fluorescent-labeled by conjugation to quantum dots. All samples were analyzed in triplicate to give 1 value per patient. Data for NCC and AQP2 were normalized by urinary creatinine.Both AQP.Serum and urine samples were prepared for analysis with a 1:25 dilution in 0.5% nitric acid  containing 0.05% Triton-X 100 (Romil) and 40 ng/ml beryllium  as an internal standard. Measurements were performed on an Agilent 7900 ICP-MS  operated in no-gas mode with the radiofrequency generator power set to 950 W giving a “cold” plasma. Lithium was measured at m/z ratio of 7 and was normalized to the beryllium internal standard at m/z 9. Calibration solutions were prepared by diluting a 1000-μg/ml lithium standard  in 2% nitric acid (Romil) to give concentrations of 0.0352, 0.141, 0.563, 2.25, 9, and 36 μmol/l. Seronorm trace elements urine quality control materials  and an in-house serum control were analyzed in each batch2, NCC3, and PGT was undertaken in healthy volunteers to establish (i) the principal urinary fraction(s) these transporters are excreted in UEVs, cell pellet, or total urine, and (ii) the optimal method of storage of urine samples for UEV analysis (fresh vs. frozen urine stored at either −20oC or −80 oC).Western blot analysis of AQP2, NCC3, and PGT was much greater in UEVs than either cell pellet or total urine, confirming that UEVs are the most appropriate fraction of urine to study  was significantly more abundant in acutely hyponatremic TIH case patients on thiazides compared to normonatremic controls on thiazides. Despite reduction in PGT in TIH case patients following cessation of thiazide and recovery to normonatremia, PGT expression was still significantly greater than in normonatremic controls off thiazides d.3 and AQP2 excretion between these patient groups. Nanoparticle tracking analysis has been used previously to quantify the abundance of UEVs containing AQP232-positive UEVs in both acutely hyponatremic and convalescent normonatremic states compared to controls  was undertaken. Patients acutely hyponatremic on thiazides with TIH displayed significantly greater phosphorylation at T55, T60, and S91 than either convalescent normonatremic TIH case patients off thiazides or normonatremic controls on thiazides .Renal endogenous lithium clearance (ELC) was lower in acutely hyponatremic TIH case patients on thiazides compared to convalescent TIH case patients on recovery to normonatremia following thiazide cessation . The ELC2 in their UEVs relative to appropriate control groups.Here we report a noninvasive method of obtaining, preserving, and analyzing urinary extracellular vesicles from volunteer patients with thiazide-induced hyponatremia who were admitted to an acute internal medicine department. In addition, analysis of distal nephron sodium, water, and prostaglandin transporters contained within the extracellular vesicles suggests a possible mechanism that may underlie the pathophysiology of TIH. These data add to what has previously been reported2, and PGT. Furthermore, we demonstrate that it is not necessary to analyze only fresh urine samples, and that samples frozen with protease inhibitor are equally suitable for use. This is important, because if analysis were limited to fresh samples, the time-consuming sample processing and immediate exosome isolation would make the study of acute medical conditions such as TIH very difficult. Because acquisition of a spot urine sample, addition of a protease inhibitor tablet, and storage in a freezer can be performed by the majority of clinical and research staff with very limited training required, such sample acquisition from acute medical patients becomes a much more feasible proposition.We have shown that the extracellular vesicles fraction is the most appropriate component of human urine with which to study the expression of NCC, AQPA priori TIH must result from excessive saliuresis and/or water reabsorption. As thiazide diuretics have not been observed to produce saliuresis with urinary sodium concentration greater than those of plasma, excess water reabsorption (or ingestion) is likely to be a fundamental part of TIH pathophysiology. Reduced renal expression of NCC and increased expression of AQP2 in acute TIH would support the hypothesis that the pathophysiology of TIH results from a combination of both of these processes. We have successfully identified both NCC1 and NCC2, NCC3 and determined the phosphorylation status of 3 key N-terminal phosphorylation sites known to regulate NCC activity from the urinary extracellular vesicles. In addition, reduced endogenous lithium clearance (ELC) in acute TIH supports increased proximal sodium reabsorption reflecting augmented distal nephron sodium loss, and so is complementary to the UEV data.The increased N-terminal phosphorylation state of NCC in acute TIH suggests some degree of physiological compensation to excessive distal nephron sodium loss, by attempting to maximally activate what NCC exists in the distal convoluted tubule. Why type 1 regulation of NCC  should be impaired in TIH but type 2 regulation  should remain intact is unclear. It is notable that thiazides inhibit NCC by binding to the cotransporter from the luminal membrane, and 1 hypothesis may be that the physical binding of thiazides to NCC induces a conformational change that targets the cotransporter for endocytosis and degradation in patients susceptible to TIH. Alternatively, saliuresis and resulting kaliuresis and trend to hypokalemia seen in acute TIH patients may drive phosphorylation and activation of remaining NCC.Thiazides reduce the ability of the late diluting segment  to generate solute-free water directly, and also act by reducing effective vascular volume and thus solute delivery from the end-proximal tubule.Measuring differences in the UEV profile of acutely hyponatremic patients may also have clinical utility as an aid to TIH diagnosis where other potential contributors to hyponatremia exist. The use of UEV analysis as a diagnostic tool would require further studies to identify whether acute differences in UEV profiling exist in other causes of hyponatremia and whether profiling of a panel of UEV markers could reliably differentiate 1 cause of hyponatremia from another.2 uptake, 1 of the most important physiological regulators of distal nephron water reabsorption.SLCO2A1 (p.A396T), the gene that encodes PGT, was also recently reported to show modest association with TIH.We chose also to study PGT because it is constitutively expressed in the collecting duct, where it mediates PGE2 could potentially identify patients at higher risk for TIH. These individuals could then be offered alternative antihypertensive therapy, for example, medications from the angiotensin-converting enzyme inhibitor or calcium channel antagonist group; or, if a diuretic is required, an alternative loop or potassium-sparing type could be selected instead of a thiazide.That differences in the extracellular vesicle profile of convalescent normonatremic TIH cases exist 2 months after thiazides have been discontinued suggests that patients who are predisposed to TIH may exhibit these urinary extracellular vesicle characteristics before they are exposed to thiazides. Although a prospective study would be required to confirm this observation, it raises the possibility that pre-prescription profiling of the relative abundance of NCC to AQP2 expression in their UEVs, which would be expected to produce excessive saliuresis and water resorption. Reduced ELC also suggests compensatory increased proximal sodium reabsorption in TIH. That differences in the renal transporters responsible for these processes can be detected in urinary extracellular vesicles both acutely and following recovery from TIH raises the possibility that such techniques may have utility in prospectively identifying those individuals at high risk for TIH and in aiding the acute diagnosis of TIH in cases that are confounded by comorbidity and/or polypharmacy.In summary, we show here that TIH patients display reduced NCC expression and increased AQPAll the authors declared no competing interests.


We present a new approach for the identification of inhibitors of phosphorylation‐dependent protein–protein interaction domains, in which phenolic fragments are adapted by in silico O‐phosphorylation before docking‐based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product‐derived phenolic fragments, which were virtually O‐phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia‐1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia‐1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small‐molecule inhibitor. Creating and searching the virtual haystack: In silico O‐phosphorylation of preselected natural product‐related fragments from the SCONP  tree, virtual screening and chemical derivatization enabled the development of Stafia‐1, the first molecule shown to inhibit the transcription factor STAT5a with selectivity over the close homologue STAT5b. Protein–protein interactions mediate most biological processes, and their functional modulation by small molecules offers vast opportunities for basic research and drug development.Phosphorylation‐dependent protein–protein interactions are mediated by the phosphorylated side chains of tyrosine, serine, and threonine residues, and play an important role in signal transduction. We recently proposed O‐phosphorylation of preselected natural products as an approach for the development of non‐peptidic and non‐reactive ligands of phosphorylation‐dependent protein–protein interactions.−1, and removal of certain reactive moieties  were selected . 1 docked into the STAT3 SH2 domain with its phosphate group in close proximity to Arg609 and Lys591  O‐phosphorylation of preselected phenolic fragments of natural products,1 in FP assays already showed a slight preference of 1 for STAT5a over STAT5b, which was lost by 4,4′′‐dichloro‐substitution  was preferable over 3,3′′‐disubstitution (compound 13) and 2,2′′‐disubstitution (compound 14). In the tetrasubstituted series 15–17, the 3,3′′,4,4′′‐substituted compound 15 was more selective for STAT5a over STAT5b than the 2,2′′,3,3′′‐substituted isomer 16 and the 3,3′′,5,5′′‐substituted isomer 17, and also more selective for STAT5a than 12. Attempts to improve STAT5a selectivity of 15 by rigidifying the alkoxy substituents (compounds 18 and 19) were not successful. In the hexasubstituted series 20–21, the 3,3′′,4,4′′,5,5′′‐substituted compound 20 was significantly more active against STAT5a than 21. Compound 20, which was synthesized in three synthetic steps  with at least 9‐fold selectivity over STAT5b  and higher selectivity against other STAT family members  maintained activity against STAT5a but reduced selectivity. Deletion of one phenyl ring of 20 caused a 5‐fold drop in activity against STAT5a (compound 23), demonstrating the necessity of the m‐terphenyl phosphate moiety for STAT5a inhibition.Dose‐dependent analysis of 20 for STAT5a over STAT5b, we used wild‐type and point mutant STAT5 proteins in FP assays. The activity of 20 against the point mutant STAT5b Gln636Pro/Met639Asn/Phe640Leu/Met644Lys/Asn664Ser/Tyr679Phe (dubbed STAT5b‐6M),m) compared to wild‐type STAT5b (37±5 % inhibition at 200 μm), and thus approximately 10‐fold lower than the activity against wild‐type STAT5a . To investigate the molecular origin of the specificity of 20 against the crossover point mutant STAT5a Trp566Arg, and found its activity to be reduced by 7‐fold  was only marginally more inhibited than wild‐type STAT5b (37±5 % inhibition at 200 μm) by 20  and STAT5b‐7M , adjacent to the SH2 domain, as the crucial determinant for selective STAT5b inhibition by small molecules.20 into the crystal structure of STAT5a20 near STAT5a Trp566  as compared to STAT5a Trp566Arg  as compared to STAT5b Arg566Trp  to binding. The remaining 2.5‐fold activity gap between the triple mutant STAT5b‐3M and the 7‐fold mutant STAT5b‐7M (Ki=21.1±4.4 μm) points towards an allosteric contribution by one or more of the remaining four STAT5a/b SH2 domain divergent amino acids, which are further removed from the putative binding site of 20.Docking of 6 Figure  B, consiced Ki=6.1±4.2 μmced Ki=6.1±4.2 μm20  was slightly less active against STAT5a than the difluoromethylene phosphonate 25 , which retained selectivity over STAT5b and other STAT proteins  and Tyr699 (STAT5b), respectively, by Bcr‐Abl. Since phosphorylation is dependent on the function of the SH2 domain, a STAT5 SH2 domain inhibitor will reduce the ability of STAT5 to be tyrosine phosphorylated by Bcr‐Abl . The use of wild‐type and point mutant STAT5 proteins demonstrated that both the SH2 domain and Trp566 in the adjacent linker domain contribute to selective recognition of Stafia‐1 by STAT5a. The cell‐permeable prodrug 27, based on the Stafia‐1‐derived monofluoromethylene phosphonate 26, inhibited tyrosine phosphorylation of STAT5a with selectivity over STAT5b in cultured human leukemia cells, and represents a valuable tool to define the non‐redundant molecular functions of the two highly homologous transcription factors in tumor cells.27, especially in direct comparison with selective inhibition of STAT5b by catechol bisphosphate‐based prodrugs such as Pomstafib‐2,In conclusion, we present docking‐based screening of in silico O‐phosphorylated natural product‐derived fragments as a novel method for identifying lead structures for the development of inhibitors of phosphorylation‐dependent protein–protein interaction domains, which are of crucial importance to cellular signaling. Whereas virtual screening of phosphonates and phosphates against a phosphorylation‐dependent protein–protein interaction domain has been reported,The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Metal exposures could possibly affect allergic responses in pregnant women, although no studies have yet shown a clear relationship between the two, and such exposures might also affect the development of allergic diseases in children.We investigated the relationship between metal concentrations in whole blood and immunoglobulin E  in 14,408 pregnant women who participated in the Japan Environment and Children’s Study. The subjects submitted self-administered questionnaires, and blood samples were collected from them twice, specifically, during the first trimester and again during the second/third trimester. Concentrations of the metals Cd, Pb, Hg, Se, and Mn, as well as serum total and allergen-specific IgEs for egg white, house dust-mites (HDM), Japanese cedar pollen (JCP), animal dander, and moth, were measured. Allergen-specific IgE(s) were divided based on concentrations <0.35 or ≥0.35 UA/mL, and the metal levels were divided into quartiles.Multivariable logistic regression analysis showed that there was a significant negative correlation between HDM- and animal dander-specific IgEs and Hg and Mn concentrations. Conversely, there was a significant positive relationship between JCP-specific IgE and Hg and Se concentrations.Metal exposures may be related to both increases and decreases in allergen-specific IgEs in pregnant women. Total IgE concentrations are known to be associated with the development of allergic symptoms and sensitizations.7 The other type involves the measurement of specific IgEs, and these data show the antibody activity against specific allergens. Recently, the immunotoxic properties of metals have been pointed out and some studies have reported that there are relationships between metal exposures and IgEs. In human studies, positive associations between exposures to mercury (Hg)/lead (Pb) and serum levels of IgEs were reported.8–10 On the other hand, other studies have reported that there were relationships between Hg, cadmium (Cd), selenium (Se), and manganese (Mn) exposures and IgEs.10–12 However, the number of study subjects ranged from about only several tens of individuals to approximately one thousand, and there have been no studies that measured both total IgE and several specific IgEs at the same time.Currently, two types of IgE information are measured clinically in common allergy diagnoses.13 Therefore, the blood Cd concentration increases throughout the pregnancy period because pregnant women tend to exhaust iron stores.13 Additionally, bones are known reservoirs for Pb.14 Pregnancy and lactation are powerful stimuli for bone resorption, and Pb that reenters circulation can raise blood Pb levels in pregnant women.14,15 In this way, although the relationships between many metal concentrations and pregnancy have not been fully clarified, metals that women have been exposed to may have different in vivo metabolic fates and effects in pregnant women than those in women who are not pregnant.Various changes occur in pregnant women compared to those who are not pregnant, and pregnant women tend to have higher metal concentrations in their bodies. For example, Cd absorption increases when iron stores are depleted.16,17 This study is a cross-sectional study and the first of its kind in Japan to use JECS data to elucidate the connection between metal concentrations in blood and IgEs.Taken together, it is very important to survey maternal metal exposures and IgE levels for both maternal and childhood health. Therefore, we have tried to clarify the relationships between metal concentrations in blood and total and specific IgEs in pregnant women who participated in the Japan Environment and Children’s Study (JECS).16,17JECS has been conducted based on the Ethical Guidelines for Epidemiological Research proposed by Japan’s Ministry of Health and Welfare . The JECS protocol was reviewed and approved by the Ministry of the Environment’s Institutional Review Board on Epidemiological Studies and by the Ethics Committees of all participating institutions. The JECS was conducted in accordance with the Helsinki Declaration and Japanese national regulations, with written informed consent obtained from all participants.16The target subjects of this study were pregnant women (median age: 31 years) who participated in the JECS. They were recruited from January 2011 through March 2014. These women lived in one of 15 JECS study regions covering a wide geographical area of Japan, and they were recruited during early pregnancy at obstetric facilities and/or local government offices.n = 895) and those on medication (n = 289). The final study population was 14,408 pregnant women  and second or third trimester (T2) at prenatal examinations or via mail. Data from the questionnaire given at T1 were used in this study because IgE levels were measured using T1 blood samples. Questionnaire data for the present study is based on the “jecs-ag-ai-20160424” dataset, which was released in June 2016 and revised in October 2016.17,18 In one round, 33 mL of blood were collected from each pregnant woman. Total IgE and allergen-specific IgE levels were then measure using T1 blood samples. Metal concentrations were measured using T2 blood samples. Blood sample data for the present study is based on the “jecs-mtl-ai-20170403” dataset, which was released in April 2017.Maternal blood samples were collected during T1 (median pregnancy week: 15) and T2 (median pregnancy week: 26) of pregnancy.19 Briefly, blood samples were collected in tubes lined with sodium ethylenediaminetetraacetic acid (EDTA) and stored at −80°C until use. A standard solution of all target elements except Hg was prepared in 0.14 M nitric acid. A standard solution of Hg was produced in a mixture of 0.056 M nitric acid, 0.5% w/v H4EDTA, and 1% v/v tetramethylammonium hydroxide (TMAH). The final concentrations of Hg, Pb, Cd, Mn, and Se in the standard solutions were 200, 200, 20, 600, and 2,000 ng/g, respectively. Internal standards  were prepared in 0.14 M nitric acid. Blood samples (200 µL) were diluted to a 1:19 ratio using a solution of 2% v/v butan-1-ol, 0.1% TMAH, 0.05% w/v polyoxyethylene (10) octylphenyl ether, and 0.05% w/v H4EDTA, and then, samples were vortex mixed prior to analysis with inductively coupled plasma mass spectrometry (ICP-MS).The method used for measurements of metal concentrations in blood has already been published in our earlier paper.20 The total concentration of the Pb isotypes is given as the sum of the concentrations of 206Pb, 207Pb, and 208Pb. To correct the spectral overlap from molybdenum oxide (95Mo16O), the concentration of 111Cd was calculated using the following equation: [Cd] = [m/z 111 concentration] − [m/z 95 concentration] × [95Mo16O generation rate]. The 95Mo16O generation rate was derived using the equation [95Mo16O generation rate] = [concentration of m/z 111 when a standard solution of 100 ppb 95Mo was analyzed]/[concentration of m/z 96 when a standard solution of 100 ppb 95Mo was analyzed].Metal levels in the blood were measured using an Agilent 7700 inductively coupled plasma mass spectrometer . Method detection limits for each analyte were calculated as described previously.Dermatophagoides pteronyssinus), Japanese cedar pollen (JCP), animal dander, and moth. The results of the ImmunoCAP system were given as exact values in UA/mL, and these data were scored using the following system of seven classes ranging from 0–6: class 0: <0.35, class 1: 0.35–0.69, class 2: 0.70–3.49, class 3: 3.50–17.49, class 4: 17.50–49.99, class 5: 50.00–99.99, and class 6: ≥100. In the results, values of 0.35 UA/mL or higher were taken as a sign of sensitization.21Serum total and allergen-specific IgE levels were analyzed using immunological assays with the ImmunoCAP system  at a clinical laboratory under contract. The following specific allergens were detected: egg white, house dust-mite  before pregnancy, allergic diseases , smoking habits, partner’s smoking habits, alcohol consumption, ownership of pets, the month of T1 blood sampling, and geographic region.The distribution of metals was skewed; therefore, natural logarithmic conversions were applied to the variables before two-group comparisons using Welch’s Participants were asked to report allergic diseases diagnosed by clinicians. For the category of smoking habits, current smokers and never-/former-smokers were categorized as “Yes” and “No,” respectively. For alcohol consumption, women who consumed alcohol during pregnancy were grouped as “Yes” and women who had never consumed or quit drinking during pregnancy were grouped as “No.” We treated smoking and alcohol consumption as categorical variables in the statistical models.st quartile = Q1, 2nd quartile = Q2, 3rd quartile = Q3, 4th quartile = Q4), and we used these sub-groups in the multivariable logistic regression analysis.We divided all subjects into four groups depending on the concentration levels of each metal, so that each sub-group consisted of close to one-fourth of the total subjects .All analyses were performed in STATA version 14 , and statistical significance was assumed when Table The concentration of Cd in the samples from those who had the low egg white-specific IgE was significantly higher than that from subjects who had high IgE values for that allergen. A similar tendency was also observed for HDM-specific IgE and Pb, as well as animal dander-specific IgE and Cd, Hg, and Se. However, the Hg and Se concentrations in samples with high JCP-specific IgE values were significantly higher than those in samples with low IgE values for the same allergen Table .P values for the trends were 0.010 and 0.006, respectively). Conversely, the OR of JCP-specific IgE increased as the Hg and Se concentrations increased   . There were significant relationships between HDM-specific IgE and Hg Q4; JCP-specific IgE and Hg Q3 and Q4; JCP-specific IgE and Se Q2, Q3, and Q4; animal dander-specific IgE and Hg Q3 and Q4; animal dander-specific IgE and Se Q3; and animal dander-specific IgE and Mn Q2 and Q3. The odds ratio (OR) of HDM- and animal dander-specific IgE decreased as the Hg concentration increased (ctively) .P = 0.002). Hg and Mn concentrations decreased significantly depending on the classes of animal dander-specific IgE . On the other hand, Hg and Se concentrations increased significantly depending on the classes of JCP-specific IgE   Table .This study demonstrated that there is a positive relationship between JCP-specific IgE and Hg and Se concentrations in the blood of pregnant women. However, HDM- and animal dander-specific IgE had a negative relationship with Hg concentrations.11 Weidinger et al reported a positive relation between Hg and total IgE but not specific IgE in children.8 In a study using human peripheral blood mononuclear cells to investigate the effects of MeHg and inorganic Hg (InHg) on immunologic dysfunction, interleukin 4 (IL-4) increased in association with both MeHg and InHg exposures, and in particular, the increases in concentrations of IL-4 were lower for MeHg than those for InHg.22 In studies using rats and mice, InHg was found to induce the activation of Th2 cells and the upregulation of IL-4, and it activated polyclonal B cells and increased the level of IgE in circulation.23–25 Although the different mechanisms of the influence of Hg exposures on total IgE and specific IgE are not known, considering human and animal studies together, increased IgE may be due to the secretion of IL-4 from Th2 cells activated by Hg exposures.Hg and Se are both taken in mainly from seafood, and since there is a high interest in Hg intake in Japan, the Japanese government has announced precautions regarding the consumption of seafood during pregnancy. There are only a few epidemiological studies that have reported on the association between Hg and IgEs in humans. Grandjean et al reported that prenatal methylmercury (MeHg) exposures were slightly associated with an increase in total IgE; however, grass-specific IgE was inversely associated with prenatal MeHg exposures in children.26 Since the average age of the participants in this study was 31 years, and 97% of the subjects were under 40 years of age, it is possible that current Hg exposures might affect the Th2 responses and increased the JCP-specific IgE in this study. In addition, this allergen is only present during a specific season. A strong increase in pollen-specific IgE was observed during the pollen season, and although it decreased once the season ended, it remained elevated for 1 year.27 Therefore, the relationship between JCP-specific IgE and Hg exposures could be explained by the direct promotion of Th2 by Hg exposures during the JCP pollen season, but it is necessary to consider other mechanisms during the non-JCP-pollen season. It has been reported that information is relayed to the immune system by IgE-switched B cells, which are not responsive to IL-4-mediated signals.27 Previous studies have shown that B cell receptor (BCR) signaling can be activated by spleen tyrosine kinase (Syk) via phosphorylation reactions in B cells.28 And Vaillant et al indicated that the BCR signaling pathway was also related with an increase in antibody production.29 When JCP enters the human body as an allergen, IgE-switched B cells may not die because of the abnormal BCR signaling pathway, and this may promote the formation of memory B cells and long-lived plasma cells, which can induce excessive IgE production resulting in an allergic response.30 McCabe et al31 reported that low levels of InHg attenuate the BCR signaling pathway, and Caruso et al32 reported that Hg alters the BCR signaling pathway, especially through inducing changes in Syk activity in experimental studies. In short, abnormalities in the BCR signaling pathway may occur in subjects who have been exposed to Hg, and consequently, IgE-switched B cells do not die and JCP-specific IgE is more likely to be produced throughout the year.In an investigation of the natural history of the allergens, an analysis of the distribution of sensitization over time revealed that JCP-specific IgE reaches a peak in humans at the age of 40 years and then decreases rapidly thereafter.33 However, Se is toxic to humans when taken in at high doses through acute inhalation or dermal contact.34–36 Hoffmann37 reported that the relationship between Se and Th2 responses is not dose-dependent. One possible effect of Se is the induction of the Th1 immune system according to the mouse model38; however, this relationship is not consistent with the results in humans. Therefore, further studies are necessary to clarify the relationship between Se exposures and allergic responses.Deficiencies of Se, which is an essential element, generally pose a risk for negative effects on the immune systems and brains of pregnant women and fetuses.39 Unlike JCP, HDM and animal dander sensitizations are prominent in children,40 and HDM tends to not become a newly sensitized antigen as a person grows to adulthood.39,40 In addition, once HDM-specific IgE responses increase in childhood, it is difficult for decreases to occur.41 Therefore, adulthood HDM and animal dander-specific IgEs do not necessarily indicate accurately sensitizations in adulthood and it was difficult to draw a clear conclusion from the results of this study. In order to clarify the relationship between HDM- and animal dander-specific IgEs and metal exposures, further studies that include metal exposures and the allergic sensitization situation during childhood will be necessary.The HDM-specific IgE had a negative relationship with Hg, and animal dander-specific IgE had a negative relationship with Hg and Mn. The mechanism of IgE sensitization to aeroallergens can change depending on the patient’s age.30,42,43 Several researchers have reported that IL-4 expression is induced in the presence of Pb and Hg in both in vivo and in vitro studies.22–24,44,45 Hg exposures were also reported to alter the phosphorylation status of Syk in the BCR signaling pathway associated with IL-4-independent IgE production in WEHI-231 cells, a murine immature B lymphoma cell line.31,32 Taken together, Hg may interact with some components involved in the pathway of IgE production, and this is followed by an increase of IgE.Based on our discussion and the results of previous research, we offer the following hypothesis on the mechanism for the upregulation of IgE production by heavy metals, which is related to allergic disease Figure . There a46 There is a notable study that showed that IL-4 can be produced under the influence of progesterone.47 Therefore, it is impossible to deny the possibility that pregnancy itself affects IgE production, and that the relationship between metal exposures and IgE might show stronger results in pregnant women compared to non-pregnant women. Regardless, a study targeting non-pregnant women is also needed in the future. Second, we could not determine the source of the exposure to the metals. According to the Japanese Ministry of Health, Labor and Welfare, however, more than 80% of Hg intake, which was significant in the present study, is derived from seafood.48 However, an investigation of whether occupational exposures are a factor is also needed. Third, because of the limited volume of blood collected from each pregnant woman, IgE and metal concentrations were measured using blood specimens collected at different times. The half-life of Hg in fish-eating humans is a few months.49 In the case of continuous exposure, a single-compartment model with a 70-day half-time predicts that the whole-body steady state .49 In the present study, the food frequency questionnaire survey was conducted twice, in T1 and T2, and only 1% of the participants changed their fish consumption between the two surveys. Thus, we assumed that Hg exposure levels used in the present analysis reflect the exposure levels in T1, when blood IgEs were measured. The half-life of other metals is about 10 years for Cd, 5 years for Pb, a few months for Se, and a month for Mn.50,51 In particular, Mn has a short half-life and the fact that the periods for Mn and IgE measurements were different might have influenced our results. Further investigations using a proper study design for such metals are warranted to confirm our findings. Fourth, Cd, Hg, and Mn concentrations in our samples were higher than those in other countries, while Se and Pb levels were similar.52–54 Therefore, it is unknown whether similar results would be obtained in other countries, and additional research is warranted.The present study has some limitations that merit discussion and further research. First, our target subject was only pregnant women. Pregnancy hormones, such as progesterone and human chorionic gonadotrophin, promote Th2 immunity.This is the first study to link metal concentrations with IgEs in pregnant Japanese women. We showed that metal exposures may be related to both increases and decreases in allergen-specific IgEs and that JCP-specific IgE, in particular, was positively related to Hg concentrations.


Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP) enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL), CYP2C9 (36.49/12.5 µg/mL), CYP1A2 (52.20/25.1 µg/mL); competitive inhibition of CYP3A4 (83.95/14.5 µg/mL) and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL). However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day), B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%). These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19. Bacopa monnieri (Linn.) Pennell (Scrophulariaceae), also known as brahmi in Ayurvedic medicine, has been used in traditional and Ayurvedic medicine for centuries as a brain tonic to enhance learning and memory and to improve concentration [B. monnieri is widely marketed and used not only in India but also in the international market. Since B. monnieri is used as a neuropharmacological agent [B. monnieri-related products by patients with mental illnesses are very high. These patients are also most likely to be prescribed therapeutic drugs, raising the potential for herb-drug interactions. ntration . These tntration ,3,4,5, antration ,7,8, whintration . A reputntration ,11, B. mal agent , the chaDrug interactions can lead to serious adverse events or decreased drug efficacy. These interactions may occur through the inhibition or induction of hepatic and intestinal drug-metabolizing enzymes  and transporters  . CYP-medet al. [The major CYPs involved in the hepatic metabolism of most drugs include CYP1A2, CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 . CYPs aret al. , and CYPet al. . B. monnieri products are classified as herbal or natural products, and in most countries, the regulatory requirements to market natural products are less stringent compared to conventional drugs because natural products are considered to be low risk products [B. monnieri that show promising results in clinical trials [B. monnieri products and the lack of information on the effect of B. monnieri extract and extract constituents on CYP enzymes warrant the study of this extract and its constituents on human CYP enzymes. In this study, B. monnieri standardized methanol extract and some of the reportedly active and commercially available constituents, including bacoside A, bacoside A3, bacopaside II, bacopaside X, bacopasaponin C and bacopaside I  CYP reactions represent total CYP activity (without any inhibition = 100%). The modulation of the CYP activity by the test compound was determined by comparing the changes from the average net signal of untreated CYP reactions with the changes observed due to the test compound. The changes were typically observed as decreases due to CYP inhibition. The test compounds that inhibit CYP enzymes caused a reduction in CYP activity and therefore generated less light/signal.The inhibitory effects of B. monnieri extract and the constituents against CYP450 were determined by evaluating the IC50 values. According to Kong et al. [50 values, as potent, if IC50 ≤ 20 μg/mL or ≤10 μM, moderate if IC50 20–100 μg/mL or 10–50 μM, or weak if IC50 ≥ 100 μg/mL or ≥50 μM. All positive controls were found to show potent CYP450 inhibition and the IC50 values were consistent with previously reported values [B. monnieri extract was found to exhibit moderate inhibition against CYP2C19, CYP2C9, CYP1A2, and CYP3A4 and weak inhibitory activities against CYP2D6 (50 = 23.67 ± 2.84 µg/mL). However, all of the constituents, bacoside A, bacoside A3, bacopaside II, bacopaside X, bacopasaponin C and bacopaside I at concentrations up to 100 µM, showed negligible inhibition towards the five CYP enzymes  and investigating the possible mode of inhibition for B. monnieri extract on the activities of human CYP1A2, CYP3A4, CYP2C9 and CYP2C19 isoforms  were studied using recombinant human CYP1A2, CYP3A4, CYP2C9, CYP2C19 and CYP2D6 enzymes. The pharmacokinetic parameters  show that B. monnieri extract most strongly inhibits CYP2C19 followed by CYP2C9, CYP1A2, and CYP3A4 and most weakly inhibits CYP2D6. However, all of the bacosides showed negligible inhibition towards all five CYP enzymes. Bacosides are dammarane-type triterpenoid saponins that have one or more sugar chains linked to a nonpolar triterpene aglycone skeleton. Due to the presence of three glycosides in the bacoside structure, these molecules have low log P values and a high number of hydrogen bond acceptors and donors. The high polarity and the low log P value of bacosides could result in low affinities to CYP active sites and negligible inhibition of the CYP isoforms. These results are similar to the CYP inhibitory activity of other triterpenoid saponins such as asiaticoside [et al. [50 values, indicating negligible or low potential for asiaticoside to modulate CYP2C9, CYP2D6 and CYP3A4 enzymatic activity. Of the seven ginsenosides tested on the catalytic activity of cDNA expressed CYPs , only one showed weak inhibition of CYP3A4 and CYP2D6. The author suggested that the tested ginsenosides are not likely to inhibit drug metabolizing enzymes and would not inhibit the metabolism of co-administered drugs that are primarily eliminated through CYP450. The above result implies that the tested bacosides are not responsible for the inhibition of the CYP isoforms by B. monnieri extract. The inhibition of the CYP enzymes by B. monnieri extract could be due to the presence of other constituents in the extract. This could include free aglycones, such as jujubogenin [The modulation of CYP activity by ticoside  and ginsticoside . Pan et  [et al.  reportedubogenin  and pseuubogenin , which aubogenin .50 values for B. monnieri extract suggest moderate inhibition of the CYPs present in the liver, a significant amount of CYPs, especially CYP3A4, are also present in the intestine. Since B. monnieri is administered orally, a high concentration of B. monnieri in the gut might cause significant inhibition of CYP3A4, CYP2C9 and CYP2C19 and reduce the activity of these enzymes to less than 10% of the total activity. Therefore, the concomitant use of B. monnieri with clinically prescribed drugs that are substrates of CYP3A4, CYP2C9, and CYP2C19 isoforms, particularly those exhibiting poor bioavailability due to extensive metabolism such as clozapine [B. monnieri as a brain tonic and the suggested use for Alzheimer’s disease (AD) [B. monnieri as an alternative medicine, and some prescription drugs that are used for AD, anxiety, depression and epilepsy are metabolized by CYP2C19, CYP2C9, CYP1A2, and CYP3A4. Although the IClozapine  and midalozapine  could caase (AD) , anxietyase (AD) , depressase (AD)  and epilase (AD) , mental B. monnieri extract inhibits CYP2C19, CYP2C9, CYP1A2, CYP3A4, patients taking B. monnieri with the drugs mentioned above could experience an increase in the plasma level of the drugs, which could result in significant adverse reactions or toxicities. CYP2C19 is involved in the metabolism of centrally active antidepressants, including citalopram, clomipramine, imipramine; anxiolytics, such as diazepam and alprazolam; and anticonvulsants, such as phenobarbital . Drugs tB. monnieri could result in clinically important herb-drug interactions in individuals who are already “poor metabolizers”. In the light of this, further investigation on the in vivo drug-herb interaction between B. monnieri extracts with the abovementioned drugs may be of importance.Furthermore, CYP2C19 and CYP2C9 exhibit genetic polymorphisms . In a ge+), glucose-6-phosphate, magnesium chloride (MgCl2) and glucose-6-phosphate dehydrogenase functioning to initiate and sustain the CYP450 reaction by maintaining a non-limiting NADPH system), reaction buffer, Luciferin Detection Reagent and Luciferin-Free Water. The membranes were prepared from baculovirus-infected insect cells and contained recombinant human CYP enzyme and P450 reductase . The negative control membranes were devoid of CYP activity. The dried powder of B. monnieri standardized methanolic extract containing 50% bacosides (batch No.: C92352/H) was purchased from Sami Labs Ltd. . Bacoside A, bacoside A3, bacopaside II, bacopaside X, bacopasaponin C and bacopaside I were purchased from Chromadex Inc. . The positive controls quinidine, ketoconazole, α-naphthoflavone, troglitazone and sulfaphenazole were purchased from Sigma Aldrich .The assay was carried out using P450-Glo™ Screening System  from Promega . The system includes a membrane preparation containing recombinant human cytochrome P450 (CYP) enzyme, negative control membranes, a luminogenic substrate appropriate for the CYP enzyme, an NADPH regeneration system  were 0.01–100 µM. The concentration of the extracts, compounds and positive controls were prepared at 4× the original concentration. All extracts and compounds were dissolved in dimethyl sulfoxide (DMSO). The organic solvent was kept below 0.25% (v/v) in the incubation mixture. First, 12.5 μL of the 4× test compounds or positive controls (appropriate for each enzyme) were added to the “treated” wells. For the “untreated”  and “minus-P450 control” wells , 12.5 μL of vehicle (1% DMSO) was added. Then, 12.5 μL of the 4× control reaction mixture  were added to the minus-P450 control wells, and 12.5 μL of the 4× reaction mixture  were added to all other wells. The plate was pre-incubated at room temperature for 10 min, and then 2× NADPH regeneration system was added to initiate the reaction. After incubation at room temperature for 30 min (45 min for CYP2D6), 50 μL of the reconstituted Luciferin Detection Reagent was added to stop the reaction and generate the luminescent signal. Before reading, the plate was incubated at room temperature for 20 min to stabilize the luminescent signal. The luminescence in all of the samples was measured using an Infinite F200 plate reader . The values were displayed as relative light units (RLU). A summary of the reaction components and the assay conditions are listed in The enzyme assay was performed in 96-well white bottom flat plates . The concentration of versus log concentration of test compounds were plotted to calculate the IC50 values. Ki values and mode of inhibition were further determined for those with IC50 less than 100 μg/mL (for extracts) or 100 μM (for active constituents). Ki and mode of inhibition were determined by incubating a series of B. monnieri extract concentration with different concentrations of respective substrates for each CYP. The percentage of CYP enzyme activity 50 values by non-linear regression analysis. The mode of inhibition was determined graphically from the Lineweaver-Burk plots. The Ki values were determined using the secondary plots constructed based on the slope of Lineweaver-Burk plots.Prism Version 5.02  software was used to calculate the ICB. monnieri standardized extract inhibited CYP enzymes, but the constituents bacoside A, bacoside A3, bacopaside II, bacopaside X, bacopasaponin C and bacopaside I showed negligible inhibition. From the IC50 and Ki values, the results indicate that B. monnieri extract moderately inhibited CYP2C19, CYP2C9, CYP1A2, and CYP3A4, but weakly inhibited CYP2D6. Competitive inhibition was observed for CYP3A4, and non-competitive inhibition was observed for CYP2C19, CYP2C9 and CYP1A2. Furthermore, at estimated gut concentrations, B. monnieri showed potent inhibitory effects towards the intestinal CYPs. Hence, the pre-systemic herb-drug interaction through intestinal CYP3A4, CYP2C9 and CYP2C19 should be considered a possibility. The compounds that are responsible for the inhibition of the CYPs remain unknown and require further work. Co-administration of B. monnieri preparations with drugs that are primarily cleared via CYP2C19, CYP2C9, CYP1A2, CYP3A4 catalyzed metabolism should be performed with caution.In summary,


Morus alba L., and showed excellent hypoglycemic effect and fewer side effects in the Phase II/III clinical trials. Thus, this paper aims to investigate the selective inhibitory effect and mechanism of SZ-A and its major active ingredients  on different α-glucosidases (α-amylase and disaccharidases) by using a combination of kinetic analysis and molecular docking approaches. From the results, SZ-A displayed a strong inhibitory effect on maltase and sucrase with an IC50 of 0.06 μg/mL and 0.03 μg/mL, respectively, which was similar to the positive control of acarbose with an IC50 of 0.07 μg/mL and 0.68 μg/mL. With regard to α-amylase, SZ-A exhibited no inhibitory activity at 100 μg/mL, while acarbose showed an obvious inhibitory effect with an IC50 of 1.74 μg/mL. The above analysis demonstrated that SZ-A could selectively inhibit disaccharidase to reduce hyperglycemia with a reversible competitive inhibition, which was primarily attributed to the three major active ingredients of SZ-A, especially 1-DNJ molecule. In the light of these findings, molecular docking study was utilized to analyze their inhibition mechanisms at molecular level. It pointed out that acarbose with a four-ring structure could perform desirable interactions with various α-glucosidases, while the three active ingredients of SZ-A, belonging to monocyclic compounds, had a high affinity to the active site of disaccharidases through forming a wide range of hydrogen bonds, whose affinity and consensus score with α-amylase was significantly lower than that of acarbose. Our study illustrates the selective inhibition mechanism of SZ-A on α-glucosidase for the first time, which is of great importance for the treatment of type 2 diabetes mellitus. α-glucosidase inhibitors (AGIs) have been an important category of oral antidiabetic drugs being widely exploited for the effective management of type 2 diabetes mellitus. However, the marketed AGIs not only inhibited the disaccharidases, but also exhibited an excessive inhibitory effect on α-amylase, resulting in undesirable gastrointestinal side effects. Compared to these agents, Ramulus Mori alkaloids (SZ-A), was a group of effective alkaloids from natural  Non-insulin-dependent diabetes mellitus  has become a serious health concern characterized by hyperglycemia due to inadequate production of insulin, causing complications such as cardiovascular diseases, retinopathy and nephropathy. Growing evidence suggests that elevated postprandial glucose is closely associated with the occurrence of DM ,2. To daMorus alba L.) have been reported to have strong inhibitory effect on α-glucosidase, which can effectively reduce postprandial hyperglycemia  denotes the concentration of substrate, V represents the enzyme reaction velocity and KTo further understand the molecular mechanism for the selective inhibition of α-glucosidase by SZ-A, molecular docking was then performed to reveal the binding modes between alkaloids and α-glucosidase. The available co-crystal structures of α-glucosidases with inhibitors were retrieved from the Research Collaboration for Structural Bioinformatics (RCSB) Protein Data Bank . To obtain sequence and structural information of α-glucosidase proteins by comparison, sequence alignment and structure superimposition were performed using the alignment/superposition tool with default parameters in MOE . 2 of crystal structures was calculated using the solvent accessibility tool with default parameters in Discovery Studio v4.0 (DS) from Accelrys  [To determine the extent of exposure to solvent of the α-glucosidase structures, the Solvent Accessible Surface (SAS) area in ÅCA, USA) .Docking calculations were carried out using the Glide program . Generally, the crystal structures of HPA (PDB code 3OLD), human NtMGAM (PDB code 3L4W), CtMGAM (PDB code 3TOP), and NtSI (PDB code 3LPP) were obtained from the RCSB Protein Data Bank. The proteins were automatically cleaned up by the preparation tools for some common problems, such as incomplete residues, the lack of hydrogens and the existence of crystallographic water . The actIn general, the docking score is used to evaluate the binding mode between the compounds and enzymes. Compared with any single scoring procedure, consensus scoring is more robust and accurate with a moderate number of scoring functions . Therefo50 values of 0.078 × 10−6 M and 0.113 × 10−6 M, respectively, even stronger than that of acarbose; (ii) 1-DNJ, FA, DAB and SZ-A exhibited no inhibitory activity on α-amylase compared with acarbose, which provided the evidence for developing an effective agent to reduce the postprandial hyperglycemia with minimal side effects; (iii) 1-DNJ, FA and DAB achieved decent consensus scores and formed favorable interactions with the active site of MGAM and SI, more so than HPA, which revealed that the selective inhibition mechanism of the major active ingredients in SZ-A on α-glucosidase at the molecular level. In addition, the molecular docking analysis validated the inhibition activity of 1-DNJ, FA and DAB on α-glucosidase, which indicated that our results were reliable. The above results indicate the unique enzymatic profile advantages of SZ-A and its major active ingredients. In the near future, there is potential for follow-up study to further investigate the possible synergistic effects and mechanisms of the different compounds in SZ-A on α-glucosidase. In conclusion, this study illustrates the selective inhibitory effect and mechanism of SZ-A on α-glucosidase and contributes in providing a reference for the clinical applications of SZ-A. To further understand the selective inhibition mechanism of SZ-A on α-glucosidase, a combination of inhibition kinetics and molecular docking simulation were conducted for the first time. The principal results showed that: (i) the main active ingredients in SZ-A  exhibited strong inhibitory activity on sucrase and maltase in competitive inhibition, among them 1-DNJ displayed the highest activity with IC


Glaciers and rivers are the main agents of mountain erosion. While in the fluvial realm empirical relationships and their mathematical description, such as the stream power law, improved the understanding of fundamental controls on landscape evolution, simple constraints on glacial topography and governing scaling relations are widely lacking. We present a steady state solution for longitudinal profiles along eroding glaciers in a coupled system that includes tectonics and climate. We combined the shallow ice approximation and a glacial erosion rule to calculate ice surface and bed topography from prescribed glacier mass balance gradient and rock uplift rate. Our approach is inspired by the classic application of the stream power law for describing a fluvial steady state but with the striking difference that, in the glacial realm, glacier mass balance is added as an altitude‐dependent variable. From our analyses we find that ice surface slope and glacial relief scale with uplift rate with scaling exponents indicating that glacial relief is less sensitive to uplift rate than relief in most fluvial landscapes. Basic scaling relations controlled by either basal sliding or internal deformation follow a power law with the exponent depending on the exponents for the glacial erosion rule and Glen's flow law. In a mixed scenario of sliding and deformation, complicated scaling relations with variable exponents emerge. Furthermore, a cutoff in glacier mass balance or cold ice in high elevations can lead to substantially larger scaling exponents which may provide an explanation for high relief in high latitudes. Glacial steady state topography is predicted based on the shallow ice approximation and a glacial erosion ruleIce thickness and surface slope in glacial topographic steady state depend on rock uplift rate and mass balanceCompared to the fluvial realm, altitude‐dependent mass balance may reduce sensitivity of glacial landscapes to rock uplift rates Our approach extends the work of Headley et al.  by using22.1Here we first follow the idea of Headley et al.  to derivU and erosion rate ė:The change of bedrock elevation over time in an active orogen is commonly described as the result of a competition between rock uplift rate ė is commonly modeled as proportional to basal sliding velocity us  is an erodibility coefficient (or erodibility) and l an exponent likely to lie between 1 and 2 ė=Kgl is only needed to calculate us in equation If rock uplift rate and erodibility are uniform under the glacier, erosion rate and hence sliding velocity must be uniform as well  via the mass balance gradient βx and in relation to the horizontal position of the equilibrium line, Ex. This is consistent with the approach of Headley et al. , sliding velocity (us), and ice thickness (H):If we further assume that the glacier is in equilibrium with climate such that, at every point along the glacier, mass outflux exactly balances mass influx plus (or minus) the ice accumulation (or ablation), ud and us can be written as a function of deformation (fd) and sliding (fs) parameters, H and ice surface slope dh/dx  of a steady state glacier longitudinal profile can finally be obtained by integrating ice surface slope and considering the ELA, E.Equation z can further be obtained by subtracting H from h. This approach produces a glacier with an ice flux that is symmetric about the ELA  only in the initial model iteration to arrive at a first guess for ice surface elevation. For this, glacier mass balance is initially assumed to vary linearly with x as defined by an arbitrary mass balance gradient βx. The elevation of the ice surface h predicted in the initial run is then used to calculate an updated glacier mass balance B fromE is the ELA and with β taken from observations g Figure a, while g Figure c. In thiPaterson . The ref2.2.1Glacier mass balance depends on altitude through a broad range of processes: the atmospheric lapse rate , which, for a given total amount of precipitation, determines the proportion falling as snow, the ice and snow ablation, the short‐wave radiation absorption, and the incoming long‐wave radiation  or a thick debris cover (γ < 1) below the ELA . From the ELA to the terminus the flux decreases more rapidly than it increases from the top to the ELA.In our glacial equilibrium approach any variation of glacier mass balance over elevation can be prescribed. We introduce two simple scenarios in addition to the case of a linear mass balance with elevation. First, mass balance gradients may differ upstream and downstream of a certain point along the glacier, here assumed to be the ELA, as defined by a glacier mass balance ratio Bm, such that mass balance is uniform above a certain elevation, according toSecond, we consider the existence of a mass balance cutoff, Bm is set to 0.4 m/year and is reached 400 m above the ELA. This leads to a reduction in glacier mass balance and flux, and, consequently, to lower ice thickness, steeper ice surface slopes, an increase in glacial steady state relief, and a decrease in glacier length. In both cases of mass balance change, maximum H, and q are still located at the ELA.In Figures 2.3Ad has been experimentally determined  and deformation becomes more and more dominant, a behavior that can be expected in glaciers. The deformation and sliding parameters are then obtained fromAbove the ELA, the change of equation . Note thequation . Howeverfd and fs lead to a strong increase in glacial relief as reduced basal sliding has to be compensated by steeper slopes  or deformation (fs≪fd) are assumed to be the dominant mechanism of ice motion in a simple glacier, which we define as temperate with constant temperature and mass balance gradient  or −0.5 (l = 2) according to Headley et al. (l = 1) or −0.2 (l = 2) in this study. This is a consequence of considering the glacier mass balance as a function of elevation instead of distance along the glacier, which leads to a feedback between ice surface elevation and glacier mass balance.Note that the scaling exponents for dy et al. . For exay et al.  report ay et al. , but −0.4.2U, Kg, β, and q in these scenarios.The basic relations between rock uplift rate, glacier mass balance, ice thickness, and ice surface slope for sliding‐ or deformation‐dominated glaciers all follow power laws. However, the scaling becomes complicated if ice sliding and deformation interact. Furthermore, scaling relations may be complicated by a break or cutoff in glacier mass balance and an elevation dependence of ice viscosity and basal sliding. Such scaling relations are difficult to report and interpret, but we suppose that a realistic scenario of a glacial equilibrium likely deviates from a simple glacier by incorporating a mix of ice deformation and sliding, a mass balance cutoff, or some temperature dependence. We thus report model implications on the related scaling of ice surface slope and ice thickness with rock uplift rate, mass balance gradient, and ice flux Figure . Scalingh/dx and U increases with U  have an ice flux pattern that is symmetric about the ELA. A nonlinear glacier mass balance over elevation as introduced in section w, along its length based on observations of alpine glaciers with a wide accumulation basin and a narrow tongue. The width is given byw0 is the width at the terminus, w1 is a scaling parameter, and k is a shape parameter. The area of the glacier can be obtained by analytically integrating width along the glacier profile:k, w0, and w1 as defined in Table Glacial equilibrium profiles derived from uniform given by6w=w0+w1xH and q are located below the ELA while glacial erosion and us are uniform.The wide accumulation area in conjunction with the narrow tongue defined by our formulation of glacier width leads to channeled ice and an increased ice thickness below the ELA which results in an increased glacier length Figure . In thisU, Kg, and β. It is difficult to argue whether this is a realistic scenario for a glacial steady state as the related hypsometry remains essentially unknown. In any case, absolute values of H and dh/dx, and thus glacier shape and topography, are affected by including glacier width and the AAR, but related scaling relations do not (fixed width) or only marginally (fixed AAR) depend on these influences for the tested parameter ranges. The only exception is a combination of a mass balance cutoff with a fixed AAR for U > 2×10−3 m/year.The way glacial relief, ice surface and bedrock slopes, and ice flux are affected by glacier width of course depends strongly on the formulation. With a fixed width, the AAR changes with rock uplift rate in our approach, if influences like temperature‐dependent sliding or a mass balance cutoff are included. In contrast, with a fixed AAR, the width of the glacier has to change with the governing parameters 6The limiting effect of a climatically induced glacial buzzsaw on the height of mountain ranges has been extensively discussed in the literature (see section 6.1U=ė), and thus,D is drainage area and i and j are constants. Note that drainage area (i) and slope (j) exponents are commonly called m and n in related literature. The scaling of slope with U is then determined by the slope exponent j fromIn defining glacial steady state topography we were inspired by the work of fluvial geomorphologists who constrained the most important long‐term controls on fluvial topography and the shape of related steady state landscapes Flint, . In a flj is still debated. In many modeling studies j≈1 is assumed  is assumed  or 0.5 (l = 2), or even higher  or ≤0.3 in case of a nonlinear glacial erosion law (l = 2);ice surface slope, glacial relief, and mean elevation increase with rock uplift rate in a glacial steady state, with characteristic scaling exponents ≤0.6 in case of a linear glacial erosion law  or ≥−0.2 in case of a non‐linear glacial erosion law (l = 2);ice thickness decreases with rock uplift rate with exponents ≥−0.4 in case of a linear glacial erosion law (ice thickness increases with glacier mass balance gradient with exponents ≤0.6;substantially higher scaling exponents of ice surface slope, glacial relief, and mean elevation with rock uplift rate are possible in case of a low glacier mass balance cutoff or reduced sliding due to cold ice;an increase in rock uplift rate raises mountain height in both fluvial and glacial steady state landscapes, but in the glacial realm glacier mass balance and hence ice flux increases with elevation, which counteracts the growth of relief and reduces the sensitivity to tectonic forcing;a glacial buzzsaw concept that conforms to the shallow ice approximation and glacial erosion rules should incorporate the coupled impact of tectonic and climatic forcing on glacial steady state topography and the resulting reduction of sensitivity to rock uplift rates in comparison to a fluvial equilibrium.We modeled the shape of glacial steady state longitudinal profiles in a coupled system in which the interaction of climate and tectonics controls glacial topography and ice thickness, based on the formulations of the shallow ice approximation and a glacial erosion law. We derived scaling relations for ice thickness and ice surface slope for sliding‐ and deformation‐controlled scenarios, as well as scenarios with a sliding‐deformation mix, and other influences such as a mass balance cutoff and temperature‐dependent ice viscosity and basal sliding properties. From these analyses we constrained the dependence of glacial relief and mean elevation on rock uplift rate and compared it to mathematical formulations of fluvial topography. Our modeling results imply thatWe elaborated a quantitative relationship between glacial landscape characteristics and tectonic and climatic controls under the assumption of topographic steady state. Further research will focus on whether glacial landscapes that correspond to our steady state solution exist on our planet and to which average climatic conditions various glaciated mountain belts under different tectonic settings may have adjusted to.


The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) stimulates gene expression via the cAMP-regulated transcriptional coactivator (CRTC) family of cAMP response element-binding protein coactivators. In the basal state, CRTCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm by 14-3-3 proteins. cAMP signaling inhibits the SIKs, leading to CRTC dephosphorylation and nuclear translocation. Here we show that although all CRTCs are regulated by SIKs, their interactions with Ser/Thr-specific protein phosphatases are distinct. CRTC1 and CRTC2 associate selectively with the calcium-dependent phosphatase calcineurin, whereas CRTC3 interacts with B55 PP2A holoenzymes via a conserved PP2A-binding region (amino acids 380–401). CRTC3-PP2A complex formation was induced by phosphorylation of CRTC3 at S391, facilitating the subsequent activation of CRTC3 by dephosphorylation at 14-3-3 binding sites. As stimulation of mitogenic pathways promoted S391 phosphorylation via the activation of ERKs and CDKs, our results demonstrate how a ubiquitous phosphatase enables cross talk between growth factor and cAMP signaling pathways at the level of a transcriptional coactivator.  •The mammalian CRTC family is distinctly regulated by protein phosphatases•CRTC1 and CRTC2 bind to calcineurin, and PP2A is recruited to CRTC3•CRTC3-PP2A complex formation is mediated by phosphorylation at S391 of CRTC3•Mitogenic signals induce S391 phosphorylation via MAPKs and CDKs Biological Sciences; Biochemistry; Molecular Biology; Cell Biology The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is a potent driver of cellular responses to hormonal and environmental cues . cAMP meUnder basal conditions 5′ AMP-activated protein kinase family members, most notably the salt-inducible kinases , sequester the three CRTCs in the cytoplasm by phosphorylation at conserved sites, which promotes phosphorylation-dependent 14-3-3 protein binding . cAMP st2+/calmodulin-dependent protein phosphatase calcineurin (CaN)  , which bin (CaN) . The mosin (CaN) , althougMultiple CRTC family members are co-expressed in mammalian tissues: CRTC1 and CRTC2 are produced in the brain  and pancBased on the importance of CRTC dephosphorylation for transcriptional activation, we systematically examined the relative association of individual CRTC family members across the mammalian complement of Ser/Thr-specific protein phosphatases by mass spectrometry. We identified selective interactions of CRTC1/2 with CaN and uncovered an unexpected role for PP2A in binding to and activating CRTC3 in response to extracellular signals. These results point to a novel role for PP2A in mediating effects of growth factor cues on the selective activation of a distinct CRTC family member.Although they are all regulated by cAMP and the SIKs, individual CRTC family members can perform unique roles A and 1B.We confirmed the mutually exclusive interactions of CRTC2 with CaN and CRTC3 with PP2A by co-immunoprecipitation (coIP) F. ExposuTo characterize the PP2A-binding region (PBR) in CRTC3, we generated serial N- and C-terminal truncations of CRTC3 and analyzed these for PP2A association by coIP . This anAlthough CRTC2 does not bind PP2A, it contains a PBR-related sequence at 425–444 D. We genThe CRTC3 PBR contains three conserved Ser/Thr residues that appear to undergo phosphorylation  Figure A. We conTo determine the importance of S391 phosphorylation for the CRTC3-PP2A interaction, we tested whether substitution of this phosphoacceptor with alanine or a phosphomimetic (Glu) alters complex formation C. In conIn a transient CRE luciferase-based assay for the quantification of cAMP-dependent gene expression , the phoPP2A has multifaceted roles in the cell cycle and is thought to function as a tumor suppressor . Various50: CDK1/2/5 = 0.2–0.8 μM, ERK1/2 = 15–30 μM) (50: ERK1/2 = 0.004/0.001 μM)  (391PLT394) containing a casein kinase 1 (CK1) consensus motif pS/Txx(x)S/T (underlined = CK1 phosphorylated residue)   and the .001 μM)  reduced .001 μM)  and mito.001 μM)  are prol= 52 μM)  also inhresidue) . In contresidue) , indicat50: CDK1/2/5 = 0.001–0.003 μM)   blocked To test the effect of ERK and cAMP signaling on endogenous S391 phosphorylation of CRTC3, we stimulated HEK 293T cells with either 12-O-tetradecanoylphorbol-13-acetate (TPA)  or Fsk C. In conFollowing exposure to TPA or nocodazole, we detected two immunoreactive bands using the S391 phospho-specific antiserum. Both bands correspond to endogenous CRTC3, as revealed in knockdown studies using CRTC3-specific short hairpin RNA (shRNAs) . In prevNR4A1 and regulator of G-protein signaling-2 (RGS2)  . The inhThe second messenger cAMP promotes cellular gene expression via the PKA-mediated phosphorylation and inhibition of the SIKs, leading to CRTC nuclear translocation upon dephosphorylation and release from 14-3-3 proteins A. Here wP219GINIF, P555NIILT and mCRTC2: P249GINIF, P614NIILT). Although PxIxIT motifs regulate the substrate affinity of CaN  forms part of an optimal ERK1/2 motif (Px[S/T]P) (RRRQPPVS391PLTL)  the S391 phosphorylation site (PVSx[S/T]P) . The PBR391PLTL) . Stimuli391PLTL) . Superim391PLTL) . Indeed,391PLTL) , which c391PLTL) . Consist391PLTL) .RRRQPPVS391PLTLSPGPE). Only B55 PP2A subunits were recovered from IPs of all CRTCs, which contain a common sequence inside their PBR-related region consisting of two hydrophobic residues flanking an SP motif (mCRTC1/2/3: PLS332PITQ/PLS434PLSL/PVS391PLTL). Although it lacks an essential glutamate at position 6, this common PBR-like motif in CRTC family members bears more similarity with the LxxIxE motif described for the B56 PP2A holoenzyme. Similar to our findings, phosphorylation of residues within the LxxIxE motif also increases B56 PP2A affinity, leading to the dephosphorylation of distal residues in relevant substrates such as Repo-Man (LSPIPE), BubR1 (LSPIIE), and RacGAP1   . Future The second messenger cAMP is traditionally thought to inhibit cell division, but in a subset of tissues, cAMP appears to cooperate with MAPK/ERK signals in promoting proliferation and differentiation . This crWe used HEK 293T cells to characterize CRTC3-PP2A complex formation and its regulation by MAPK- and CDK-mediated phosphorylation. Although this approach was valuable in characterizing these molecular components and their downstream effects on CRTC3 activation, the relevant extracellular signals that act upstream to trigger the interaction of CRTC3 with PP2A remain relatively unknown. Our studies with brown pre-adipocytes support a potential role for ERK in mediating effects of β-adrenergic signals on recruitment of PP2A and on the subsequent activation of CRTC3. Future studies with phospho (S391)-specific antiserum should reveal the physiological contexts in which this site is phosphorylated, and mutant mice expressing phospho-S391 defective CRTC3 should provide insight into the physiological importance of this modification.All methods can be found in the accompanying


Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia.To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling.We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming.Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD. LRRK2omplexes . Among aomplexes , with thomplexes . The G20omplexes –9 and Raomplexes , 11.LRRK2 is expressed in several brain regions, including the substantia nigra pars compacta, striatum, hippocampus, cortex, and olfactory bulb , 13. As In its inactive form, PKA is a tetrameric enzyme composed of a R subunit dimer and two catalytic (C) subunits. In the absence of cAMP, a dimer of R subunits binds and suppresses the activity of two C subunits. Conversely, the cooperative binding of cAMP to the R subunits causes a conformational change that leads to the activation of PKA and consequent phosphorylation of its targets . TypicalBuilding on previous observations reported by us  and otheTaken together, our results indicate that LRRK2 kinase activity is a crucial regulator of PKA signaling in microglia and propose PDE4 as a novel LRRK2 effector protein in these cells.All animal procedures were carried out in strict accordance with the recommendations issued in the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) for animals housed at NIH and for the European Community Council Directive 2010/63/UE for animals housed at the University of Padova. The protocols were approved by the Institutional Animal Care and Use Committees of the US National Institute on Aging  and by the Ethics Committee of the University of Padova (Project ID 1041/2016-PR), respectively.g, and re-suspended in DMEM-F12, supplemented with 10% FBS, 2 mM glutamine, 2 mM sodium pyruvate (Sigma Aldrich), penicillin, and streptomycin. Cell suspension obtained from three brains was plated on poly-L-lysine -coated T-75 flask. After 4 days, the medium was replaced and the mixed glial culture was maintained until day 14. At 12 days, microglia cells were isolated from the mixed culture by shaking for 4 h at 160 rpm, and the purity of the obtained culture was verified by double immunofluorescence with mouse anti-CD11b  for microglia cells and with rabbit anti-GFAP (DAKO #Z0334) for astrocytes. The amount of astrocyte contaminants was negligible.BV2 cells were cultured in RPMI-40 medium (Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin, and streptomycin. HEK293T cells were cultured in Dulbecco modified Eagle medium  supplemented with 10% FBS, penicillin, and streptomycin. Primary microglia cells were derived from postnatal days 1–4 (P1–4) LRRK2 wild-type and G2019S knock-in (KI) mice as recently described . Specifi2 controlled atmosphere.All cells were maintained at 37 °C in a 5% COHEK293T cell transfections were performed using polyethylenimine (Polysciences) following the manufacturer’s recommendations. Eukaryotic expression constructs of 3xFlag-tagged LRRK2 WT and G2019S, green fluorescence protein (GFP)-tagged PKA RIIβ and GFP empty vector, generated as described previously , were usDuring treatments, BV2 cells were cultured in medium containing 1% FBS. LRRK2 inhibitor GSK2578215A  and forskolin (Sigma Aldrich) were used at 2 μM and 30 μM, respectively, for 90 min. PDE4 inhibitor rolipram (Tocris Bioscience) was used at 10 μM for 30 min. Dimethyl sulfoxide (DMSO) was used as control.Escherichia coli, BL21(DE3), with strongly reduced endotoxicity [Human α-Syn pffs were generated from recombinant α-Syn produced by a lipid A mutant of toxicity . After pBV2 cells and mouse brains were solubilized as recently described . ProteinHEK293T cells were harvested at 48 h post-transfection. For co-IP assays, cells were lysed in lysis buffer  and incubated with anti-Flag M2 affinity gel (Sigma Aldrich) overnight. For pull-down assays, tagged proteins were purified using GFP-trap resin (CromoTek) and then incubated for 2 h with cell lysates containing endogenous prey protein. Immuno-complexes were washed three times with lysis buffer supplemented with 0.25 M NaCl, resuspended in sample buffer, and then subjected to immunoblotting analysis.https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_truseq/truseqstrandedtotalrna/truseq-stranded-total-rna-sample-prep-ls-euc-ltf-15031060-e.pdf). Before sequencing, cDNA libraries were quantified by digital PCR using the ddPCR Library quantification kit (Biorad). Subsequently, cDNA libraries were multiplexed with four samples per pool and 7 pM of each pool hybridized to a flow cell following cluster generation using an Illumina cluster station. Libraries were sequenced on Illumina HiSeq2500 (Illumina) to generate ~ 35 million of 100-bp single-end reads per library.After shaking, isolated microglia cells were seeded for 2 days and then collected for RNA extraction, which was performed as previously described . RNA quacAMP levels were quantified by using cAMP Elisa kit  according to the manufacturer’s protocol. Specifically, BV2 cells were first treated with 2 μM LRRK2 GSK inhibitor or DMSO for 90 min and then with 30 μM forskolin for 15 min to enhance cAMP contents. Five independent samples for each condition, assayed in two technical replicates, were used for the analysis.t test or one-sample t test, while for multiple comparisons by one-way ANOVA with Tukey’s post hoc test. Data were analyzed using Prism (GraphPad).All quantitative data are expressed as mean ± SEM and represent at least three independent sets of experiments. Statistical significance of differences between two groups was assessed by unpaired G2019S KI mice exhibited 50% reduced S337 phosphorylation compared to WT mice  . AccumulWe previously demonstrated that loss of LRRK2 or inhibition of its kinase activity causes a reduction of NF-κB-dependent transcription of inflammatory genes after LPS or α-Syn pffs priming, which is mediated by enhanced PKA activity and consequent increased NF-κB p50 inhibitory signaling . In addiAccumulating evidence indicates a functional interaction between LRRK2 and PKA, although the precise molecular mechanisms of this cross-talk still need to be elucidated . In thisThe available literature supports the notion that the functional interaction between LRRK2 and PKA may be bidirectional. PKA can act upstream of LRRK2 through direct phosphorylation of distinct LRRK2 serine residues , 32, butG2019S KI mice exhibit increased phosphorylation of RIIβ compared to WT mice, suggesting that LRRK2 G2019S plays an inhibitory effect on PKA activation. In support of these results, LRRK2G2019S KI mice displayed reduction of PKA-mediated NF-κB p50 phosphorylation, a well-established PKA phosphorylation target [In this study, to investigate the molecular mechanism underlying LRRK2-PKA cross-talk in microglia, we started exploring LRRK2-PKA RIIβ interaction and RIIβ phosphorylation state as readouts of PKA activation state in relation to LRRK2. Auto-phosphorylation of S114-RIIβ, or S99-RIIα, by C subunits controls the interaction between RII and C subunits  and the n target , whereasTo gain more insights into the molecular mechanism of this regulation, we initially tested the hypothesis that LRRK2 modulates PKA activity through direct phosphorylation of RII subunits, but we did not find any convincing evidence from in vitro kinase assays (unpublished observations). PKA is part of a multifunctional complex composed of different signaling molecules, including PPs and PDEs, which are essential for compartmentalization and regulation of PKA activation state . In partG2019S KI mice exhibit increased inflammation compared to WT microglia upon stimulation with α-Syn pffs. These observations suggest that LRRK2 G2019S, as well as all other pathological mutations that confer increased kinase activity, favors the transition of microglia toward a pro-inflammatory state, which, in turn, may result in an exacerbated inflammation and neurodegeneration in LRRK2-related PD patients. Supporting this hypothesis, LRRK2 G2019S carriers exhibit higher levels of peripheral NF-κB-dependent inflammatory cytokines compared to control subjects [Taken together, our results provide a further evidence supporting a PKA-LRRK2 axis in microglia cells, with LRRK2 kinase controlling PKA activation through PDE4; however, the molecular mechanism underlying LRRK2-PDE4 functional interaction remains to be explored. In agreement with reduced NF-κB p50 phosphorylation in the presence of hyperactive LRRK2, we found that primary microglia isolated from LRRK2subjects , and ratsubjects .Overall, our findings indicate that LRRK2 kinase activity is a negative regulator of PKA signaling and suggest that PDE4 may be a novel LRRK2 effector protein in microglia. Future studies directed at understanding LRRK2-dependent regulation of PDE4 will offer a more defined scenario of LRRK2 biology and pathobiology in these cells. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.