We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl− currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I− > Cl− > gluconate− permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl− conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl The biophysical and pharmacological profile of this current is remarkably similar in different cell types. Its activation threshold is around pH 5.0, it is strongly outwardly rectifying, shows time-dependent activation at constant positive membrane potentials (Vmem), maintains an ion permeability sequence of SCN− > I− > NO3− > Br− > Cl−, and it is blocked by a wide range of unspecific Cl− channel blockers such as DIDS, NBBP, niflumic acid, or phloretin [Strong extracellular acidification induces an acid-sensitive outwardly rectifying (ASOR) anion current/channel in many cell types, which is also known as PAORAC , or IClhloretin ,12,13,14−) current, which is also known as volume-regulated anion current (VRAC), swelling-dependent Cl− current (IClswell), volume-sensitive organic osmolyte and anion current (VSOAC), or regulatory volume decrease current (RVDC). The current is slowly activated by osmotic cell swelling or early in apoptosis and it is substantially involved in regulatory volume decrease (RVD) and apoptotic volume decrease (AVD), respectively, and cell volume-related processes, like cell proliferation, migration, and phagocytosis [Some of these properties, like outward rectification, ion permeability sequence, and blocker sensitivity, are similar to the ubiquitously expressed and extensively characterized volume-sensitive outwardly rectifying (VSOR) anion . The cells undergo morphological changes by retracting their processes and increasing cell size upon the detection of neuronal damage, nervous system dysfunctions, or brain lesions. In the activated state, they migrate to the site of brain injury and phagocytose affected cells and cell fragments . Migrati process ,20. In m process ,26,27,28 process ,25. More process , showingA role of the ASOR current in these processes is elusive. In the case of ischemia, inflammation, tumor, or traumata, when pH regulating mechanisms fail to maintain a constant pH, the acidotoxic environment causes the swelling of neurons and glial cells, which poses a challenge to the cell volume regulatory mechanisms ,31,32,33n = 6), 5.0, 4.0, and 3.0 (n = 12) with half-maximal current activation at a pH of ~5.3.The baseline whole-cell currents were low in patch clamp recordings that started under isotonic conditions and an extracellular pH of 7.2. Reducing the pH to 5.0 or lower elicited a strongly outwardly rectifying anion current. Activation was slower and gradual at pH 5.0 and was steeper at lower pH values . The mean ASOR current amplitudes recorded at pH 4.5 were 2.30 ± 0.17 nA (I1) and 2.50 ± 0.19 nA (I2) at +100 mV and −0.39 ± 0.08 nA (I1) and −0.11 ± 0.02 nA (I2) at −100 mV (n = 18) (p < 0.0001) and current inactivation at −100 mV (p < 0.001) at constant holding potentials  in The pH dependency of activation and current kinetics were identical to the acid-sensitive outwardly rectifying (ASOR) Clll types ,12,13,14(n = 18) d,g and ttentials g. This i− current, which we have previously characterized in BV-2 cells [1 (1.84 ± 0.19 nA) than at I2 (1.62 ± 0.14 nA) , which indicated time-dependent inactivation at constant positive holding potentials, which is phenotypical to this current (2/I1 ratio < 1.0 (1) only slightly, though significantly (p < 0.01), declined over time to −0.63 ± 0.07 nA at I2 , while VSOR currents were higher in the inward direction (p < 0.05) (The volume-sensitive outwardly rectifying (VSOR) Cl-2 cells ,27, deve-2 cells ,23  g.− in the bath solution was replaced with equimolar concentrations of iodide (I−) or gluconate to determine the relative anion permeabilities of the ASOR current. In the presence of I− and gluconate, the reversal potential (Erev) shifted by −9.94 ± 1.16 mV (p < 0.001) and 29.77 ± 0.10 mV (p < 0.01), respectively. Relative permeabilities, as calculated from the shifts in Erev while using the Goldman-Hodgkin-Katz equation, were I− : Cl− : gluconate− = 1.53 ± 0.07 : 1 : 0.25 ± 0.01  Cl− currents [n = 6; p < 0.001) and 12.0 ± 1.5% , inward currents at −100 mV to 48.7 ± 16.5% , and 50.9 ± 4.1%  of control values, respectively. In contrast to DIDS, the currents showed significantly different sensitivities to DCPIB and tamoxifen. The ASOR current was far less sensitive with an inhibition to 76.9 ± 4.7%  and 66.2 ± 15.4% (n = 6) while the VSOR current was strongly inhibited by 10 µM DCPIB to 5.8 ± 2.4%  and 9.7 ± 3.6%  at +100 and −100 mV. Moreover, the ASOR current was completely insensitive to 10 µM tamoxifen, a concentration which caused an almost complete block of the VSOR outward and inward current to 3.4 ± 1.3%  and 4.8 ± 1.9%  of control, respectively.We used the Clcurrents . As show+-free, as well as hypertonic (350 mOsm/kg) conditions. The higher outward current amplitude (p < 0.001) under hypertonicity can be explained by the larger step in pH to 3.5 as compared to the controls (pH 4.5). Further, the current could be activated in the ruptured patch clamp configuration and in the presence of the membrane permeable Ca2+ chelator BAPTA-AM, which indicated that an intact intracellular compartment and intracellular Ca2+ ions are not prerequisite for current activation.The acid-sensitive current could be readily activated under extracellular Nai) over time using the membrane permeable pH-sensitive dye BCECF-AM to test how far extracellular acidification transfers to the intracellular milieu. The pHi significantly dropped to pH 6.8–6.5 within 20 min. and remained relatively stable 0.6–2 pH units above the prevailing extracellular pH over the next 55 min. after reducing the extracellular pH to 6.0, 5.0, or 4.5 . Warming to 37 °C increased the outward current by 28.2 ± 2.8% , while the inward current was not significantly affected . The VSOR outward and inward currents were diminished by 31.6 ± 2.1% and 36.4 ± 3.6%, respectively  at 4 °C , but also of the inward current at −100 mV by 79.7 ± 18.5% .We performed recordings at room temperature (22 °C) and after superfusion with a 4 °C and 37 °C bath solution to test for temperature effects on ASOR and VSOR currents. As shown in  at 4 °C c,d, whicn = 5–7). We applied 500-ms voltage steps from 0 mV to +100 and −100 mV at these time points to analyze the current inactivation/activation behavior at constant holding potentials  at +100 and −100 mV, respectively  and −1.79 ± 0.15 nA  at −100 mV upon simultaneous acidification to pH 4.5. As visible in the traces (3) and (3.1), the whole-cell Cl− current showed distinctive kinetics at +100 mV, with an initial inactivation phase followed by current facilitation over time characteristic for the ASOR current , which indicated that the volume-sensitive current inactivated under lasting acidic conditions. Stepping back from pH 4.5 to 7.2 under continued hypotonicity caused rapid ASOR deactivation (time point (5)), while the VSOR current slowly recovered from acidic inhibition to reach amplitudes of 2.07 ± 0.17 nA at +100 mV and −1.10 ± 0.15 nA (n = 5) at −100 mV at time point (6).Switching from isotonic to hypotonic conditions at pH 7.2 was followed by slow VSOR current activation from the baseline current levels to 1.79 ± 0.08 nA and −0.70 ± 0.06 nA  under pH 4.5, and 2.13 ± 0.39 nA and −0.30 ± 0.14 nA  under additional hypotonic exposure. This implies that VSOR current activation is prevented at low pH. However, stepping back to pH 7.2 under persisting hypotonicity again caused rapid ASOR current inactivation and slow VSOR current activation to 1.68 ± 0.46 nA and −0.77 ± 0.43 nA (n = 3) at +100 and −100 mV with a characteristic time course and current phenotype . mem recording before, during, and after extracellular acidification to pH 4.5. In this experiment, with a latency of ~5 min., acidification led to a stable and reversible depolarization to ~−20 mV. We briefly switched from current to voltage clamp and applied voltage ramps to test whether the ASOR current was activated before, during, and after the acidity-induced depolarization (mem under pH 7.4 was −66.13 ± 5.42 mV (n = 8) . After switching back to pH 7.4 Vmem repolarized to −55.45 ± 9.61 mV .Next, we tested how far ASOR activation affects the cell membrane potential (Vrization b. The av (n = 8) c. Under − channel blockers DIDS or DCPIB. We observed a 10–15% cell shrinkage from ~2 400 to ~2 100 fl over 60 min.  response to counteract osmotic cell swelling ,16,22,23− current in BV-2 microglial cells for the first time and describe its interrelation with the volume-sensitive (swelling-activated) outwardly rectifying (VSOR) Cl− current. ASOR currents have been described in diverse cell types, such as epithelial cells, myocytes, osteoclasts, chondrocytes, neuronal cells, and erythrocytes [− currents are remarkably similar. The current is activated by acidification to pH ≤ 5.0, is outwardly rectifying, and shows facilitation over time at constant positive holding potentials. In BV-2 cells, we recorded maximum ASOR current amplitudes between pH 4.0 and 3.0. The current declined again after reaching peak amplitudes at pH values < 3.0, similar to as described in HEK293 cells [− > Cl− > gluconate− anion permeability sequence, which is typical for volume-sensitive- and Ca2+-activated Cl− channels [− ≥ I− > gluconate−) has been reported for ASOR channels in mouse osteoclasts [via a low-pH pipette solution does not activate the current, which suggests that the proton binding sites are located at the extracellular face of the plasma membrane, and that the intracellular pH does not apparently affect ASOR channel activation. This is supported by the finding that the endogenous ASOR current in HEK293 can be readily activated when using an intracellular solution that contains 100 mM HEPES [In this paper, we characterize an endogenous acid-sensitive outwardly rectifying (ASOR) Clhrocytes ,14,34,3593 cells ,27. Addichannels ,22,23, ichannels ,11,14. A− transport blockers, such as DIDS, NPPB, niflumic acid, or 1,9-dideoxyforskolin, inhibits the ASOR currents [50 values from 0.12 µM in HeLa cells [− conductances in a cell system. For DCPIB and tamoxifen, which are regarded as more specific for VSOR currents [Like VSOR currents, a wide range of structurally unrelated Clcurrents ,11,12,34currents , we foun− currents, which is surprising, given the plethora of literature on these currents. Similar to the VSOR current, the acid-sensitive current was augmented by warming and diminished by cooling. However, in contrast to the volume-sensitive current, warming only increased the outward acid-sensitive current, while the inward currents were largely unaffected. Higher outward ASOR currents at 37 °C as compared to room temperature have also been reported in HeLa cells [ASOR and VSOR currents in BV-2 cells are both temperature-sensitive, however in different ways. VSOR current amplitudes were significantly higher at 37 °C than at room temperature and cooling to 4 °C had the opposite effect. To our best knowledge, this is the first report of the temperature effects on volume-sensitive ClLa cells  and mousLa cells .− of ~−40 mV while assuming the complete equilibration of the cytoplasmic Cl− concentration with the pipette solution during the recordings. This implies that, besides ASOR currents, the activation of acid-sensitive cation conductances is likely to contribute to the depolarization. e.g., functional expression of both acid-sensing ion channels (ASICSs) [Extracellular acidification to pH 4.5 caused a strong depolarization of the cell membrane potential to ~−28 mV, which is above the calculated reversal potential of Cl(ASICSs)  and acid(ASICSs)  has been− current immediately after switching from pH 4.5 to 7.4 under continued hypotonicity, since the volume-sensitive current first needs to be reactivated from acidic inhibition. The partial inhibition of VSOR currents and accelerated inactivation at positive potentials under less acidic conditions (pH 5.0 and higher) has previously been shown in Xenopus oocytes [Including our present work, only a few studies have been performed to investigate the interrelation between ASOR and VSOR channels/currents by combining the hypotonic- and acidic challenge. There is a controversial discussion as to whether the same or different channel entities underlie both ASOR and VSOR currents. While Nobles et al.  postulat oocytes , C6 glio oocytes , BC3H1 m oocytes , mouse n oocytes , bovine  oocytes , and end oocytes . Under w2+-activated Cl− currents (CaCCs) and show certain phenotypical similarities to ASOR currents  that form the ASOR channel. In recent years, the pannexin-related leucine-rich repeats containing 8A (LRRC8A/SWELL1) protein and its paralogs have been identified as essential VSOR channel components ,45. It htentials ), resulttentials . ASOR cutentials ,7,11, theoclasts  and HEK2eoclasts , others eoclasts ,11,35. Icidic pH , where tnel pore ,50, whic+ ions [via proton channels [Functions of the ASOR current, or the consequences of its activation under physiological or pathophysiological conditions, are elusive when considering the massive extracellular acidification that was required for the channels to open. Importantly, Sato-Numata et al. observed a shift in the half-maximum activation pH value of the ASOR current from 4.9 at 25 °C to 5.4 at 37 °C in mouse cortical neurons . The aut+ ions ,7,8. A fchannels . It is rchannels . Proton − ions and/or organic osmolytes might underlie the loss in cell volume under control conditions. That DIDS was without effect might be due to its voltage-dependent inhibition with lower blocking efficiency when compared to DCPIB at normal resting membrane potentials. Under isotonic conditions and pH 4.5, the cells showed a more inconsistent behavior. Although, on average, the cell volume was relatively constant, we observed ~5% cell swelling in two out of three experiments. We assume that this is the manifestation of impaired cell volume regulation and acidotoxic/necrotic volume increase, similar as that previously described in microglial cells [− current activation and the activation of acid-sensitive cation channels, such as ASICs or TRPM7 channels. Cell swelling under acidic conditions was enhanced by DIDS, which could be due to the inhibition of the residual volume-sensitive Cl− current or other DIDS-sensitive transport mechanisms. This contrasts findings in HeLa cells and cortical neurons, where acidosis-induced swelling was prevented by DIDS and phloretin [− due to Cl− current activation, the relative contribution of Cl− ions to the osmotic driving force for water efflux would be reduced and the efflux of organic osmolytes would remain as the main driving force for the RVD. However, if ASOR channels, in contrast to VSOR channels, were impermeable for organic osmolytes, ASOR current activation would have the same effect as pharmacological inhibition of the VSOR current. In any case, our data imply that cell volume homeostasis in microglial cells is massively impaired under acidic conditions, and that ASOR channels cannot substitute for VSOR channel function in cell volume regulation.VSOR currents play a key role during RVD in counteracting osmotic cell swelling ,16,22,23al cells , neuronsal cells , and HeLhloretin . DCPIB pp.a. grade. DIDS, nigericin, and tamoxifen were purchased from Sigma-Aldrich-Merck , DCPIB from Tocris-biotechne , and BCECF-AM from Merck-Calbiochem . Stock solutions of nigericin (5 mg/mL), tamoxifen (40 mM), and DCPIB (100 mM) were prepared in ethanol. DIDS and BCECF/AM were dissolved in dimethyl sulfoxide (DMSO) to give stock solutions of 100 and 10 mM, respectively. The stocks were stored in aliquots at 20 °C until use.All the salts and chemicals were 3  containing 10% fetal bovine serum  and 1% antibiotic-antimycotic solution  at 37 °C, 5% CO2, and 95% air (standard culture conditions). The subcultures were established once a week by trypsin/EDTA  treatment.The immortal cell line BV-2  was grown in RPMI 1640 Medium with stable glutamine and 2.0 g/L NaHCOd-lysine (PDL)-coated coverslips (12 mm diameter) and cultured for at least 24 h under standard conditions. The coverslips were transferred to an RC-25 recording chamber  and then mounted on a Nikon Eclipse TE2000-U inverted microscope. Unless otherwise stated, the experiments were performed at room temperature in the whole-cell perforated patch clamp configuration while using amphotericin B. The recordings were started when the serial resistance was <30 MΩ for the perforated configuration and <10 MΩ if the ruptured configuration was applied. Patch electrode resistances were 4–9 MΩ. Data were recorded while using an EPC-10 amplifier controlled by PatchMaster software . Cell membrane potential (Vmem) recordings were performed in the zero-current clamp mode. The intracellular (pipette) solution contained (in mM): 70 K2SO4, 10 NaCl, 10 KCl, 4 MgCl2, 2 CaCl2, 5 HEPES free acid, 10 EGTA , and 130 µM amphotericin B. The isotonic extracellular control solution contained (in mM): 140 NaCl, 5.6 KCl, 2.5 CaCl2, 1.5 MgCl2, 10 HEPES free acid, 4.5 glucose, and 5 mannitol . The voltage clamp recordings of ASOR and VSOR currents were performed under symmetrical intra- and extracellular Cl− conditions. The isotonic extracellular control solution consisted of (in mM): 100 NaCl, 2.5 CaCl2, 2.5 MgCl2, 10 HEPES free acid, and 80 mannitol . Mannitol was omitted to obtain a hypotonic (220 mOsm/kg) solution for VSOR activation. To assess pH dependencies, the extracellular solution was titrated to pH values ranging from 3.0 to 5.0. The pH was adjusted with HCl into a range of ± 0.05 from the intended values immediately prior to use. The pipette solution contained (in mM): 100 CsCl, 5 MgCl2, 10 HEPES free acid, 11 EGTA, 60 raffinose, 2 Mg-ATP , and 130 µM amphotericin B. The pipette solution was titrated to pH 4.5 (HCl) for recoding the effect of an intracellular acidification. The currents were monitored in response to voltage ramps  and voltage steps  from −100 to +100 mV. The holding potential between the ramps/steps was 0 mV to desensitize the voltage-activated currents. Bath solution exchange was performed with a valve-controlled gravity-driven perfusion system  at a flow rate of 3–5 mL/min. Osmolalities of intra- and extracellular solutions were measured while using a Wescor® vapor pressure osmometer.The BV-2 cells were seeded on 0.01% poly-g and the supernatants were discarded. The cell pellet was re-suspended in 20 ml of an extracellular solution containing (in mM) 140 NaCl, 5.6 KCl, 2.5 CaCl2, 1.5 MgCl2, 10 HEPES free acid (FA), 4.5 glucose, and 5 mannitol immediately before the first measurement (time point 0) (300 mOsm/kg), in which the pH was adjusted to pH 7.4, 6.0 or 4.5 with NaOH or HCl. For the pH 4.5 solutions, MES hydrate was used instead of HEPES free acid. The pH of the solutions was titrated into a range of ± 0.05 from the intended value, immediately before use. The hypotonic extracellular solution (220 mOsm/kg) was obtained by a reduction of NaCl to 100 mM. Blockers (DIDS or DCPIB) were added to the samples, as indicated. The mean cell volumes (MCV in fl) in the different samples were alternately measured every 5 min. over 60 min. on a Beckman Coulter Z2 particle counter . Between two measurements, the aperture tube was rinsed with a solution of the same pH as in the following sample to minimize the pH alterations by liquid carryover. The Coulter method is based on measuring changes in electrical resistance produced by nonconductive particles that were suspended in an electrolyte solution (Coulter method). Calibration for particle size was done while using 10-µm Flow-Check fluorospheres (Beckman Coulter). The data were analyzed with the Multisizer Software (Beckman Coulter) while using a 600-fl cutoff to exclude cell debris.The BV-2 cells were harvested by Trypsin/EDTA after growing for 4–5 days under standard conditions. The cell suspension was split into aliquots to measure the effect of extracellular pH. Aliquots were centrifuged for 5 min. at 200× 5 cells/mL and kept under standard conditions for 24–48 h. The cells were loaded with 5 µM of the membrane permeable pH-sensitive dye BCECF-AM for 30 min. at 37 °C in serum-free medium. After the removal of the loading medium 100 µL of extracellular solution containing (in mM) 140 NaCl, 5.6 KCl, 2.5 CaCl2, 1.5 MgCl2, 10 HEPES free acid, 4.5 glucose, and 5 mannitol (300 mOsm/kg) with pH being adjusted to 7.4, 6.0, 5.0, or 4.5 were added into the wells in quadruplicates. The pH of the solutions was titrated into a range of ± 0.05 from the intended value immediately before use. The fluorescence measurements (bottom readings) were performed at 37 °C in a humidity cassette on a Spark 20 M multimode plate reader . BCECF was alternately excited at 440 and 490 nm while using the built-in monochromator and emission was measured at 535 nm  every 5 min. over 75 min. Readings from wells containing non-BCECF-loaded cells were used for background subtraction. 490/440 nm ratios calculated from the corrected fluorescence intensity values were converted to absolute pH while using the high K+/nigericin calibration method .Cells were seeded in 96-well black, clear bottom microplates at a density of 1.5 × 10n ≥ 3). The solvent control samples were included in all experimental series. Statistical analysis was carried out using Student’s t-test, Wilcoxon test, or one or two-way ANOVA with Dunnett’s, Tukey’s, or Bonferroni’s tests for multiple comparisons, as applicable. Means were considered to be significantly different at p-values < 0.05 . Data were analyzed and plotted while using GraphPad Prism 7  or Igor Pro 6 . Data are expressed as means ± standard error of the means (SEM) of at least three independent biological replicates (−) current in BV-2 microglial cells, which shares characteristics with ASOR currents in other cell types, including its sensitivity to extracellular but not intracellular acidification, pronounced outward rectification, anion permeability sequence, and pharmacological profile. Our data add to a better understanding of the electrophysiological properties of microglial cells in general and the activation behavior of ASOR channels. We compared the properties of the acid-sensitive current with those of the volume-sensitive outwardly rectifying (VSOR) Cl− current and then worked out an interrelation between these two conductances and possible functional consequences of this interrelation for cell volume homeostasis. We find a short-term coexistence and superimposition of ASOR and VSOR currents under simultaneous acidic and hypotonic conditions, but the ASOR current dominates in the longer term, while the VSOR current gets inactivated. Importantly, we show that the cell volume regulatory ability of BV-2 cells is severely impeded under acidic conditions and conclude that ASOR channels cannot fulfill a function in cell volume regulation. Acidosis is a hallmark of pathophysiological events, like inflammation, stroke, or ischemia. Migration and phagocytosis in the microglial cells closely related to cell volume regulatory processes. Under acidotoxic conditions in the brain, ASOR current activation could therefore affect microglial function.We describe an endogenous acid-sensitive outwardly rectifying (ASOR) anion (Cl


A recently reported rapid potentiation of NMDA receptors by Group I metabotropic glutamate receptors (mGluRIs) via a Homer protein link is distinct from the classical, relatively slow inhibitory G-protein-associated signaling triggered by mGluRI activation. The relationship between these two mechanisms remains unknown. Here, we focused on the mGluRI-dependent modulation of NMDAR response in hippocampal dentate gyrus granule cells and cerebellar granule cells of C57BL6-J mice and found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. At a shorter time interval (units of millisecond), the Homer-mediated signal from mGluRIs prevails, causing upregulation of NMDAR function, in both dentate gyrus granule cells and cerebellar granule cells. Our results shed light on the possible mechanisms of anti-schizophrenia drugs that disrupt Homer-containing protein link.SIGNIFICANCE STATEMENT Here we study modulation of NMDA receptors triggered by activation of metabotropic glutamate receptors Group I via two distinct pathways: classical G-protein signaling system and newly discovered high-speed modulatory mechanism associated with Homer-protein-containing direct molecular link. We found that these two contrasting mechanisms overlap competitively on the time scale from hundreds of milliseconds to seconds, with the net effect depending on the cell type. We have also found that both crosstalk mechanisms cause significant changes in synaptic strength and plasticity. Our results resolve an apparent discrepancy between earlier studies that demonstrated contradictive effects of Homer-containing protein link disruption on NMDA receptor signaling. On top of that, our data provide a plausible explanation for unclear action mechanisms of anti-schizophrenia drugs. This siproteins , enablinThe second, more recently discovered, mechanism of mGluRI-NMDAR crosstalk is signal transfer via the Homer proteins. Homer protein family contains a C-terminal coiled-coil dimerization domain (“long” Homers), except for Homer1a and Ania-3 (“short” Homers), which lack this domain and, as a result, are unable to perform a scaffolding role linking other proteins. Homer proteins bind to the mGluRIs , and to Xenopus oocytes) , mGluRI activation in cerebellar granule cells (CGCs) potentiates NMDARs in intact cells but has no effect when the Homer molecular interlink has been disrupted by Homer1a overexpression . An earloocytes)  and in boocytes)  and hippoocytes) .It has been widely accepted that NMDAR hypofunction is one of the key factors provoking schizophrenia development . AnotherHere, we aimed to resolve these apparent functional discrepancies and to clarify the interaction of overlapping signals delivered through (1) rapid Homer-mediated and (2) slower G-protein-mediated pathways of mGluRI-NMDAR crosstalk. We hypothesized that mGluRIs, when activated, could modulate NMDARs simultaneously via the two pathways, Homer- and G-protein-controlled. The pathway that prevails is dependent on the time scale of activation: the fast Homer-transduced effect is more prominent at short-term intervals after mGluRI activation, whereas the slower, G-protein-delivered effect has an advantage on long-term intervals. To test the hypothesis, we set out to investigate the mGluRI-NMDAR interaction in two distinct cell types, where earlier studies demonstrated different long-term effects of mGluRIs on NMDAR response: potentiation   and supp4 cells/ml on glass coverslips that had been coated with poly-D-lysine  and Laminin protein . Cultures were maintained in a humidified incubator at 37°C, <5% CO2.Cultures were generated from E17.5, C57BL6-J mice. Pregnant mother was killed with cervical dislocation according to UK Animals (Scientific Procedures) Act 1986 Schedule 1. Embryos were removed by caesarean section and decapitated in Hanks Balanced Salt Solution  containing 5% penicillin-streptomycin  on ice. Skulls were removed and brain tissue dissected under a light microscope where the cerebellum were separated followed by hippocampi removal from the embryo forebrain. Tissue was then exposed to enzymatic digestion using Papain  following mechanical dissociation in complete DMEM  containing 10% FBS  and 5% penicillin-streptomycin. Tissue was washed with DMEM and pelleted twice before being resuspended in complete Neurobasal media  containing 10% B27 , 5% PenStrep and 0.25% L-gluatamine . Cells were plated at a density of 2–5 × 10DIV7 with pRK5-Homer1a overexpression vector and mCherry fluorescent marker vector  using Lipofectamine 2000 ; 250 ng DNA per vector was added to Optimem and then incubated with Lipofectamine at room temperature before being dripped onto the cultures and left to recover for 5 d before recordings were taken.Granule cells were transfected at m): 119 NaCl, 2.5 KCl, 1.3 Na2SO4, 2.5 CaCl2, 26.2 NaHCO3, 1 NaH2PO4, 22 glucose and was continuously gassed with 95% O2/5% CO2, pH 7.35 (290–298 mOsm). The intracellular pipette solution for voltage-clamp experiments contained the following (in mm): 120.5 CsCl, 10 KOH-HEPES, 2 EGTA, 8 NaCl, 5 QX-314 Br− salt, 2 Na-ATP, 0.3 Na-GTP. For current-clamp recordings, intracellular pipette solution contained the following (in mm): 126 K-gluconate, 4 NaCl, 5 HEPES, 15 glucose, 1 K2SO4 · 7H2O, 2 BAPTA, 3 Na-ATP. pH was adjusted to 7.2 and osmolarity adjusted to 295 mOsm.Visualized patch-clamp recordings from cultured granule cells were performed using an infrared differential interference contrast imaging system. The perfusion solution contained the following  neurons with short dendrites , and thehold − 70 mV). Solution exchange experiments were performed as described in our previously published protocol (Outside-out patches and cell membrane bags containing intact nucleus and cytoplasm (nucleated patches [NPs]) were pulled from DGCs and CGCs, and recordings were performed in voltage-clamp mode (Vprotocol . Brieflym), NBQX (20 μm), strychnine (1 μm), and CGP-55845 (1 μm). Recordings were performed at 32°C–34°C; the patch pipette resistance was 3–7 mΩ, depending on particular experimental conditions. Series resistance was monitored throughout experiments using a 5 mV step command; cells with unstable series resistance (>25 mΩ) or unstable holding current were rejected.Whole-cell EPSPs were measured in current-clamp mode in the presence of the following set of ligands: picrotoxin (50 μP) in control and after series of paired stimuli, when G-protein signaling cascade and/or Homer protein interlink were interrupted. To allow registration of both increase and decrease of P, in the beginning of the experiment, stimulation was adjusted to generate P in an interval 25% < P < 75%. If under control conditions EPSC component had P out of this interval, the component was not used in further statistical calculations.To assess modulation of synaptic efficacy in polysynaptic signaling pathways, we used an experimental approach tested on hippocampal cell cultures earlier . Brieflyem 580 nm). An experimental test of calbindin-D28k presence was done with a method described previously (em 510 nm). Hereupon coverslips were placed to 4% PFA for 12 h at room temperature and then incubated at 4°C for 24 h with monoclonal mouse anti-calbindin-D28k antibodies (1:1000) in TBS with 0.3% Triton X-100. Next, biotin-conjugated sheep anti-mouse antibodies (1:200) and streptavidin-conjugated Cy5 (1:300) were applied sequentially overnight; then Cy5 fluorescence imaged (λem 670 nm).DIV11–14 mCherry-positive cells were selected for tests of Homer1a-overexpression effects using induced fluorescence (λeviously . To do tRecordings were obtained using a MultiClamp 700B amplifier (Molecular Devices), filtered at 4–8 kHz, digitized at 10 kHz, and stored on a PC. pClamp/Clampfit 10x software (Molecular Devices) was used for data storage and off-line analysis.m each) at outside-out and nucleated patches evoked macroscopic responses, where “peak response” was obtained as a difference between baseline (average for 50 ms before application of NMDAR ligands) and maximum evoked current; “stable” or “equilibrated” response was obtained as a difference between baseline and stable current generated at 200–800 ms interval ∼4 s after start of continuous application of NMDAR ligands.Activation of NMDARs with high concentrations of NMDA and glycine (50 μm each) at outside-out and nucleated patches evoked single-channel openings to three conductance levels: 51.9 ± 7.8 pS, 37.2 ± 6.4 pS, and 22.1 ± 8.3 pS. The larger conductance level contributed 91.9% of the single-channel current, whereas medium and low subconductance levels contributed 6.2% and 1.9% of the current, respectively; thus, the larger main conductance level was used when (possible) changes of single-channel conductance were compared under different experimental conditions. Because it was virtually impossible to determine accurately the number of channels in a nucleated patch, the open probability was obtained as the total open probability of N channels (NPo), calculated as the proportion of the channel total open time to the duration of recording. Because NPo in individual patches widely varied (from 0.02 to 0.2), for statistical calculations we used values normalized to control (NPo generated by NMDA+Gly in corresponding patch). With the low (1 μm) NMDA and glycine concentrations used in this study, the majority of channel events were single-level events. In a case where there were multiple levels of channel openings, only levels with the highest conductance was analyzed. This prevented us from overestimation of NPo increase because in multichannel patch increased NPo would be accompanied by increase of proportion of multilevel events. To inspect the stationarity of channel activities, conductance, NPo, and the open time were followed in control experiments for up to 15 min; no time-dependent alterations were detected.Activation of NMDARs with low concentration of NMDA and glycine  to activate only NMDARs; second, 50 μm NMDA + 50 μm Gly + 50 μm DHPG to activate NMDARs and mGluRIs; being compared with response generated by NMDARs only, this should reveal mGluRIs modulatory effect on NMDARs; third, 50 μm NMDA + 50 μm Gly + inverse agonists of mGluRIs: 100 nm FTIDC and 1 μm Fenobam (FB); comparison to “NMDARs only” response should reveal whether mGluRIs modulate NMDARs due to spontaneous activation. Each solution was applied for 4 s, as this time was enough to stabilize NMDAR response amplitude. This protocol was repeated on intact cells and cells overexpressing Homer1a with standard intracellular solution and intracellular solution containing PeTX (1 μg/ml), which allowed us to register four modes of mGluRI-NMDAR crosstalk: mode i, intact cell, standard internal solution: both Homer- and G-protein signaling pathways are active; mode ii, intact cell, internal solution with PeTX: only Homer signaling pathway is active; mode iii, Homer1a-overexpressing cell, standard internal solution: only G-protein signaling pathway is active; mode iv, Homer1a-overexpressing cell, internal solution with PeTX: both signaling pathways are blocked. To account for individual patch characteristics, for statistical calculations, the response amplitudes were normalized to the amplitude of response generated by the application of 50 μm NMDA + 50 μm Gly.First, we tested the mGluRI-NMDAR interaction on nucleated membrane patches where solutions containing mGluRI and NMDAR ligands were exchanged on the same patch, with registration of stable response amplitude. Three combinations of ligands were applied at the same patch sequentially: first, 50 μp = 0.036, n = 9; mode ii, p = 0.034, n = 6; mode iii, p = 0.004 . The normalized mGluRI-modulated response in mode iii was also significantly higher than that of mode i: 1.66 ± 0.13 versus 1.3 ± 0.094, p = 0.044 .Nucleated patches from DGCs, subjected to this experimental protocol, demonstrated a significant impact of mGluRIs on NMDAR function delivered through both Homer- and G-protein signaling chains. In contrast, the application of mGluRI inverse agonists, as in all further experiments, did not cause any significant modulation of effect generated by NMDA+Gly . Normalip = 0.016 ; in all other cases, no significant difference was observed (C).When a similar experiment was repeated on nucleated patches from CGCs , only G-observed C.As a control, we tested this experimental protocol on outside-out patches excised from DGCs and CGCs, where both Homer- and G-protein signaling chains were supposed to be destroyed. As expected, in this experiment, any kind of pharmacological manipulations did not exert any significant effect .m) and Gly (1 μm) to make single-channel openings visible. Channel conductance, average open time, and opening probability were used as quantitative characteristics of the effect of mGluRIs . However, data on opening probability demonstrated a significant upregulation of NMDAR function in i–iii modes, when normalized to control: mode i, 1.52 ± 0.13 when mGluRIs were activated versus 1.12 ± 0.11 when mGluRIs were blocked; mode ii, 1.26 ± 0.1 versus 1.03 ± 0.03; mode iii, 1.65 ± 0.2 versus 1.06 ± 0.11; mode iv, 1.01 ± 0.07 versus 0.95 ± 0.15. Significance of difference from unity when mGluRIs were activated for modes i–iii: p = 0.002, p = 0.029, and p = 0.013, respectively . In mode iv, no significant difference from unity was observed (C).In DGCs, the average open time (13.7 ± 2.6 ms) and single-channel conductance (52.1 ± 2.4 pS) were indistinguishable between recordings in modes i–iv C. Howeveobserved C.C): mode i, 1.1 ± 0.15 when mGluRIs were activated versus 1.07 ± 0.1 when mGluRIs were blocked; mode ii, 1.31 ± 0.18 versus 1.23 ± 0.19; mode iii, 0.83 ± 0.06 versus 0.93 ± 0.1; mode iv, 0.93 ± 0.06 versus 0.96 ± 0.12. Significance of difference from unity when mGluRIs were activated for modes ii and iii: p = 0.042 and p = 0.037, respectively .In CGCs, similar to DGCs, the disruption of G-protein- and Homer-containing mGluRI-NMDAR crosstalk did not result in significant changes of average open time (11.9 ± 3.2 ms) and single-channel conductance (51.6 ± 3.7 pS) . HoweverFig. 5After experiments on long intervals, where Homer- and G-protein-delivered effects overlapped, we tested mGluRI-NMDAR modulation at short intervals (units of milliseconds). We presumed that, under these conditions, the G-protein-delivered effects might not have enough time to fully develop, as was observed previously .p = 0.003 and p = 0.004, respectively (n = 6). However, Homer1a overexpression prevented such a potentiation: mode iii, 3.68 ± 2.01% versus 1.87 ± 2.54%; mode iv, 1.54 ± 1.94% vs −0.02 ± 1.66% . The same experiment on CGCs . In all recordings, the maximum potentiation effect of mGluRIs developed within the 5 ms time window after the application of ligands  application of agonists, that the activation of mGluRIs has increased peak amplitudes of NMDAR response independently of PeTX presence in the internal solution. For DGCs : mode i, on CGCs : mode i, ligands , 7.Next, we asked whether and to what extent Homer- and G-protein-mediated mGluRI-NMDAR signaling pathways regulate the generation of neuronal APs. To clarify this, we performed whole-cell recordings from neurons in dispersed cell culture, where APs were evoked by consequent 300 ms applications of the same set of solutions, as in the experiments on membrane patches . To quanp = 0.044, p = 0.047, and p = 0.032, respectively . When Homer- and G-protein pathways were blocked simultaneously in mode iv, the firing frequency observed with DHPG and FTIDC+FB in the applied solution became indistinguishable from unity : p = 0.144 and p = 0.426 for DHPG and FTIDC+FB, respectively .Here we found that in DGCs blocking of each signaling pathway leads to a significant reduction of APR compared with control value: mode i, control, 1.82 ± 0.19 when mGluRIs were activated versus 1.08 ± 0.14 when mGluRIs were blocked; mode ii, 1.32 ± 0.1 versus 1.04 ± 0.13; mode iii, 1.52 ± 0.09 versus 1.1 ± 0.11; mode iv, 1.17 ± 0.18 versus 1.02 ± 0.12. Significance of difference from control when mGluRIs were activated in modes ii–iv: p = 0.042 and p = 0.021, respectively . As in DGCs, under mode iv, APR generated with DHPG and FTIDC+FB was indistinguishable from unity: p = 0.35 and p = 0.08, respectively .In contrast, in CGCs, only Homer1a overexpression triggered significant mGluRI-mediated modulatory effects on APR compared with control: mode i, control, 1.15 ± 0.17 when mGluRIs were activated versus 0.92 ± 0.13 when mGluRIs were blocked; mode ii, 1.47 ± 0.14 versus 1.08 ± 0.12; mode iii, 0.81 ± 0.08 versus 0.95 ± 0.14; mode iv, 1.05 ± 0.11 versus 1.16 ± 0.14. APR in mode iii was significantly lower compared with control and to unity: P) remained stable for each EPSC component observed in recorded cell. We thus used the EPSC profile as a tool for quantitative measurement of synaptic efficacy . To examine synaptic plasticity in recorded network, we applied a train of 50 paired-pulse stimuli with 50 ms interpulse interval at 1 Hz. After that, we monitored changes in P  of preexisting EPSC components, which reflect remodeling of signaling pathways . Recorded neurons held in modes i–iv allowed assessment of the input of two types of mGluRI-NMDAR crosstalk into modulation of synaptic plasticity.As the next step, we set out to clarify the role of two types of mGluRI-NMDAR crosstalk in synaptic transmission and induction of synaptic plasticity. To comprehend this, we used networks of cultured neurons where two polysynaptically connected cells were patched simultaneously. Current injection into one cell (held in current-clamp mode) generated AP with the subsequent poly-component EPSCs recorded from another cell (held in voltage-clamp mode). We interpreted each EPSC component as a signal through separate transmission pathway with specific delay. Under the low-frequency stimulation (1 current injection per 15 s), the probability of occurrence . In modes i—iii, block of mGluRIs caused significant reduction of ΔP compared with readout when mGluRIs were active in the same mode: p = 0.021, p = 0.031, and p = 0.036, respectively .We found, in DGCs, that blocking of each mGluRI-NMDAR crosstalk pathway induces a significant lowering of ΔP compared with control, unlike the block of G-proteins chains (mode ii) and of both signaling pathways (mode iv): mode i, control: 0.26 ± 0.013 when mGluRIs were activated versus 0.18 ± 0.03 when mGluRIs were blocked; mode ii: 0.19 ± 0.011 versus 0.14 ± 0.013; mode iii: 0.21 ± 0.013 versus 0.16 ± 0.02; mode iv: 0.12 ± 0.022 versus 0.12 ± 0.02. For comparisons of modes ii and iv: to control when mGluRIs activated . As in DGCs, in modes i–iii, block of mGluRIs caused significant reduction of ΔP compared with readout when mGluRIs were active: p = 0.018, p = 0.045, and p = 0.019, respectively .In turn, in CGCs blocking of Homer pathway (mode iii) did not trigger significant change of ΔP values obtained when mGluRIs were blocked under any experimental mode (i–iv), from ΔP obtained when mGluRIs were activated, but both mGluRI-NMDAR signaling pathways were blocked (iv): p > 0.05 for all comparisons, n = 7 (DGCs) and n = 8 (CGCs), Student's t test (C).For both DGCs and CGCs, we did not observe significant difference of Δs t test C.P of particular components, but without substantial variation of their amplitude. In DGCs under mode i, amplitude of the first peak after the stimulation train was changed by 17 ± 22% compared with pretrain value; under mode ii, by −11 ± 19%; under mode iii, by +21 ± 18%; under mode iv, by 7 ± 16% . In CGCs under mode i, first peak amplitude was changed by 14 ± 9%; under mode ii, by −8 ± 13%; under mode iii, by −4 ± 11%; under mode iv, by 9 ± 8% .As a general rule, alterations of postsynaptic current profile displayed changes in m APV) rather than of mGluRIs. We found that pharmacological silencing of NMDARs induces a significant decrease of ΔP values . For DGCs, mode i: 0.075 ± 0.023 when NMDARs were blocked versus 0.23 ± 0.033 when NMDARs were active; mode ii: 0.067 ± 0.018 versus 0.16 ± 0.039; mode iii: 0.049 ± 0.02 versus 0.18 ± 0.022; mode iv: 0.035 ± 0.014 versus 0.11 ± 0.014. For comparisons “APV-active NMDARs”: p = 0.0032, p = 0.0067, p = 0.0054, and p = 0.0086 for modes i–iv, respectively . For CGCs, mode i: 0.058 ± 0.02 when NMDARs were blocked versus 0.24 ± 0.42 when NMDARs were active; mode ii: 0.61 ± 0.022 versus 0.17 ± 0.021; mode iii: 0.048 ± 0.011 versus 0.15 ± 0.019; mode iv: 0.046 ± 0.011 versus 0.09 ± 0.024. For comparisons “APV- active NMDARs”: p = 0.0028, p = 0.0073, p = 0.0062, and p = 0.023 for modes i–iv, respectively .Next, to quantify the role of NMDARs in synaptic plasticity and signaling pathway remodeling, we repeated the same experimental protocol, but with pharmacological block of NMDARs . To do this, we patched cultured cells, preselected on visual criteria, with pipette solution containing GFP. If cell electrophysiological parameters were in predefined intervals , we performed an accurate withdrawal of patch pipette and captured an image of GFP fluorescence. Afterward, the coverslip with cell culture underwent immunostaining with Cy5 fluorescent dye for the protein calbindin-D28k, which is a characteristic DGC marker . This implies the competitive nature of the two, albeit unidirectional, signals. In contrast, in CGCs, only the G-protein pathway, being activated alone in Homer1a-overexpressing cells, induced a significant downregulation of NMDAR effect, thus resembling previous observations , which implies the lack of significant input from other mechanisms of mGluRI-NMDAR crosstalk. Next, we observed the effect of DHPG in nucleated patches  (D). Therefore, we found that G-protein-controlled and Homer-controlled signaling pathways between mGluRI and NMDAR play a significant role in synaptic plasticity and efficacy.NMDARs are widely recognized to play a pivotal role in long-term synaptic plasticity in the CNS . In our stimuli) D. TherefP), which is consistent with earlier observations of involvement of synaptic NMDARs into long-term plasticity . This may reflect involvement of mGluRI- and NMDAR-independent mechanisms, such as modification of GABA-ergic conductance . Modulation of EPSC profiles without significant impact on response amplitudes suggests that the chosen pattern of stimulation had caused variation of synaptic strength at remote synaptic connections, thus changing the probability of a signal transmission by different pathways connecting stimulated and recorded cell, with variable interplay between these pathways. This type of modification is consistent with a paradigm of distributed storage and representation of information in neural networks  is due to the overlap of Homer- and G-protein-delivered signals of opposite sign, rather than the result of, for instance, the majority of mGluRIs in the active state (due to spontaneous activation) before application of DHPG.Constitutive agonist-independent activity of G-protein-coupled receptors was repeatedly detected in various experimental setups and may substantially impact cell functioning and intercellular signaling . HoweverFig. 8Therefore, in our study, we found the functional mechanism of mGluRI-NMDAR interaction in DGCs and CGCs to be as follows : mGluRIsC) due to higher mGluRI-triggered potentiation delivered through a G-protein chain, rather than through a competing Homer interlink . This suggests a plausible mechanistic explanation for haloperidol and clozapine's anti-schizophrenia effects associated with the upregulation of Homer1a synthesis (The hippocampus has been recognized as a key brain area in schizophrenia development , associaFig. 1nterlink C, 4C, 8Eynthesis .


Dyeing of polyester fabrics with thienobenzochromene disperse dyes under conventional and microwave heating conditions was studied in order to determine whether microwave heating could be used to enhance the dyeability of polyester fabrics. Fastness properties of the dyed samples were measured. All samples dyed with or without microwave heating displayed excellent washing and perspiration fastness. The biological activities of the synthesized dyes against Gram positive bacteria, Gram negative bacteria, yeast and fungus were also evaluated.  The energy of microwave photons is quite low compared with the chemical bond energies and, thus, microwaves do not directly affect the molecular structure of a compound and they do not change the electronic configuration of atoms ,2. When H-benzo[h]chromene-3-carbonitrile 1 with elemental sulfur in presence of morpholine as basic catalyst in a microwave oven as an energy source at 130 °C for 5 min, thienobenzochromene 3 is produced in 93% yield , using the high temperature (HT) dyeing method at 130 °C. Orange to violet color shades were obtained (*L); chroma (*C); hue angle (h) from 0 to 360°; *a, whose value represents the degree of redness (positive) and greenness (negative); and *b, whose value represents the degree of yellowness (positive) and blueness (negative). Hue h is one of the three basic characters for color. The h values in 6e as a violet color. In general, the color hues of the disperse dye 6e on the polyester fabric shifted to the bluish directions; this was indicated by the negative value of *b = −14.73 and −16.78 (yellow–blue axis). The positive values of *a and *b indicated that the color hues of the disperse dyes 6a–d on the polyester fabric shifted in the reddish direction.Disperse dyes obtained . The dyeobtained , and the6a–e are darker , slightly less red , less yellow  and slightly less saturated  than the conventional samples. The K/S values are ranged from 7.03 to 39.47 and 6.89 to 23.60 for the microwave dyed fabric and the conventional dyed fabric, respectively, All of these results indicate that the microwave dyed samples are better than the conventional samples in a very short dyeing time, These results in this study support the findings of the other researchers, like Oner et al. [1]benzopyran-4-one (6a). Brown crystals (81%), mp >300 °C (lit. . Anal. Calcd for C21H13N3O2S: C, 67.91; H, 3.52, N, 11.31; S, 8.63. Found C, 67.96; H, 3.23; N, 11.06; S, 8.50%.°C -thieno[1]benzopyran-4-one (6b). Orange powder (75%), IR (KBr): 3397 and 3275 (NH2), 1702 (CO) cm−1; 1H-NMR (DMSO-d6): δ = 2.39 , 7.34 , 7.65-7.71 , 7.91 , 8.01 , 8.34 , 8.88 . MS (EI) m/z = 385 , HRMS: m/z (EI) for C22H15N3O2S; calcd. 385.0871; found: 385.0871.3-Amino-1-(4-methoxyphenyl)-thieno[1]benzopyran-4-one (6c). Deep orange powder (70%), mp 283–284 °C. IR (KBr): 3392 and 3277 (NH2), 1708 (CO) cm−1. 1H-NMR (DMSO-6d): δ = 3.56 , 7.02 , 7.62–7.80 , 7.92 , 8.25 , 8.74 . MS (EI) m/z = 401 . HRMS: m/z (EI) for C22H15N3O3S; calcd. 401.0821; found: 401.0821.3-Amino-1-(4-chlorophenylazo)thieno[1]benzopyran-4-one (6d). Orange crystals (78%), mp. >300 °C (lit. [1]benzopyran-4-one (6e). Violet crystals (74%), mp. > 300 °C (lit. . The samples were heated in this solution for 30 min. at 80 °C and then thoroughly washed and air-dried.The dye baths were prepared from the dye (2% weight of fabric) to a final liquor of 50:1, w/w. The pH value of the bath was adjusted to 4.5–5 with acetic acid (10%) in the presence of a 1:1 ratio of the dispersing agent . The temperature was raised to 130 °C in focused microwave oven  and dyeing continued for 20 min. After dyeing, the fabrics were thoroughly washed and then subjected to a surface reduction clearing [(2 g NaOH + 2 g sodium hydrosulphite)/L]. The samples were heated in this solution for 30 min. At 80 °C and then thoroughly washed and air-dried.K/S values, were determined by applying the Kubelka-Mink equation as follows:R = decimal fraction of the reflectance of the dyed fabric; oR = decimal fraction of the reflectance of the undyed fabric; K = absorption coefficient; S = scattering coefficient.The colorimetric parameters of the dyed polyester fabrics  were detAfter washing using 2 g/L of the nonionic detergent Hostapal CV at 80 °C for 15 min, the dyed fabrics were tested by using standard methods . A speciThe samples were prepared by stitching a piece of dyed polyester fabric between two pieces of cotton and wool fabrics, all of equal length, and then immersed in the acid or alkaline solution for 30 min. The staining on the undyed adjacent fabrics was assessed according to the following gray scale: 1—poor, 2—fair, 3—moderate, 4—good, 5—excellent. The acid solution (pH = 4.5) contains sodium chloride (10 g/L), sodium dihydrogen orthophosphate (1 g/L) and histidine monohydrochloride (0.25 g/L). The alkaline solution (pH = 9.5) contains sodium chloride (10 g/L), disodium orthophosphate (1 g/L) and histidine monohydrochloride (0.25 g/L).Bacillus subtilis (Gram positive bacterium), Escherichia coli (Gram negative bacterim), Saccharomyces cerevisiae (yeast) and Asparigillus niger (fungus) were used in the test. An aliquot of 0.1 mL of each bacterial strain was inoculated and spread on nutrient agar (NA) while 0.1 mL of each yeast and filamentous fungi were spread on potato dextrose agar (PDA). The inoculated plates were supplied with 100 µL of each of the tested dyes with a total final concentration of 100 mg mL−1. The dyes were included in 4 mm wells produced by sterile cork borer. The NA plates were incubated at 37 °C for 24 h while PDA plates were incubated at 25 °C for 24–48 h. For reference drugs 100 µL with a total final concentration of 100 mg mL−1 of flumox  was used as antibacterial drug reference also scored against yeast. The antimicrobial activities of some disperse dyes were tested using the agar-well diffusion technique  against In summary, a series of disperse dyes were synthesized based on the arylazothienobenzochromene moiety. The dyes produced in this manner were then applied to polyester fabrics using both conventional and microwave heating dyeing methods at 130 °C. The fabrics dyed by both methods displayed excellent fastness levels to washing and perspiration. Finally, the biological activities of the synthesized disperse dyes against Gram positive bacteria, Gram negative bacteria, yeast and fungus were discussed.


Poion alpinae pasture, and subsequently a nutritionally poor Seslerion caeruleae pasture. In milk, the highest concentrate level reduced linolenic acid and total polyunsaturated fatty acids, while the pasture type influenced the monounsaturated fatty acids. In cheeses, these differences were markedly reduced. It is well-known that milk and cheese derived from grazing animals are beneficial for human health. Grazing dairy cows produce milk with high levels of unsaturated fatty acids and conjugated linoleic acid, which are able to reduce cardiovascular diseases and have some anticancer properties. The aim of this trial was to assess the effect of pasture type and concentrate supplementation levels on the fatty acid composition of milk and cheese obtained during summer grazing on mountain pasture. Seventy-two dairy cows, supplemented with 3.0 kg/head/d or 1.5 kg/head/d of energetic concentrate feed in the diet, grazed on a nutritionally rich Poion alpinae alliance pasture (PAST1), and subsequently they grazed on a Seslerion caeruleae alliance pasture (PAST2) for 10 d each. In the last three days of each experimental period, milk samples were collected immediately before each cheese-making event. Cheese samples were collected from each cheese loaf after 60 d of ripening. LOW showed higher isoFA, FA intermediates of the ruminal biohydrogenation, C18:3 c9,c12,c15, and total polyunsaturated fatty acid (PUFA) levels than HIGH. The pasture type had a more limited effect on FA composition of milk than concentrate level and was mainly related to monounsaturated fatty acids (MUFA), which were higher in PAST1 than PAST2 (p < 0.05). In cheeses, these differences were reduced. The phytanic acid and phytanic isomer ratio (SRR/RRR) in milk were not affected either by supplement level (p > 0.05) or by type of pasture (p > 0.05). Increasing the concentrate offered to dairy cows from 1.5 to 3.0 kg/d did not markedly influence the level of PUFA in cheeses produced during summer grazing on high mountain pasture.The aim of this trial was to assess the effect of pasture type and concentrate supplementation on the fatty acids (FA) composition of milk and cheese obtained during summer grazing on mountain pasture. Seventy-two Italian Simmental dairy cows were assigned to two groups that differed by the amount of concentrate supplementation: 3.0 kg/head/d (HIGH) vs. 1.5 kg/head/d (LOW). The dairy cows grazed on a  In Europe, from 2010 to 2013, the area of permanent grassland and meadows was reduced by 2%, while the number of holdings with permanent grassland and meadows was reduced by 9% . Indeed,In mountain areas, dairy cows are kept indoors all year with the exception of the summer months, when the cows are allowed to graze. During the grazing season, dairy cows are usually moved through different pastures to take full advantage of the available territory following the vegetation gradient and the so-called ‘vertical transhumance’ animal management strategy. Moreover, the use of pasture is considered as a tool to increase the added value of summer dairy products obtained from mountain farms ,8. IndeeThis study aimed to assess the effect of pasture type and concentrate supplementation on the FA composition of milk and cheese obtained during summer grazing. These findings could be useful to enhance the summer dairy products and to reduce the abandonment of farms in mountain area.The trial was carried out in accordance with EU Directive 2010/63/EU; it complied with the Italian legislation on animal care , and adhered to the internal rules of the University of Udine.Poion alpinae alliance pasture, 1500 m asl; PAST1) for 10 d, and after another adaptation period of two weeks, they grazed on a nutritionally poor pasture  for 10 d. PAST 1 was mainly composed of 66.5% Poaceae and Cyperaceae, 7.4% Ranunculaceae, and 2.8% Fabaceae, while PAST2 was mainly composed of 61.8% Poaceae and Cyperaceae, 8.5% Asteraceae, 5.4% Fabaceae, and 3.3% Rosaceae. Both pastures were grazed at the flowering stage of Poaceae with shepherd-guided grazing. Concentrate was offered to animals twice daily in the milking parlor. More details are reported in Bovolenta et al. [Seventy-two Italian Simmental dairy cows grazing day and night on high mountain pasture  were assigned to two homogeneous experimental groups according to their productive performance recorded after a preliminary period of two weeks for milk yield (16.9 ± 3.3 kg/d), stage of lactation (192.8 ± 64.0 days in milk (DIM)), fat (3.94 ± 0.25%), protein (3.29 ± 0.14%), lactose (4.71 ± 0.12%), and somatic cell count (SCC) . The groups differed for amount of concentrate supplementation: 3.0 kg/head/d (HIGH) vs. 1.5 kg/head/d (LOW). The concentrate was mainly based on maize, barley, beet pulp, and soy, and its chemical composition and fatty acid profile are reported in a et al. . For thra et al. , which ia et al. , in the The herbage and concentrate samples were dried in a forced draft oven at 65 °C for 48 h and analyzed for crude protein (CP) and ether extract (EE) according to Association of Official Agricultural Chemists (AOAC) , and for2  + 3 [On milk samples, the following determinations were carried out: fat, protein, lactose , urea 22, and so000) + 3 . The cheese samples were analyzed for fat and protein according to AOAC . v/v) [Fatty acids of herbage and concentrate samples were determined as methyl esters, extracted by chloroform:methanol  , and estv/v) . Fatty aFat was extracted from milk following the method described by Povolo et al.  in orderEither way, the lipids were then esterificated in accordance with the method described by Bannon et al. , with moFatty acid methyl esters were detected using a GCMS Saturn 2100T  equipped with an HP-88  capillary column . Split injection (1 µl) was adopted (ratio 1:50) and helium was used as a carrier gas at a flow rate of 1.2 mL/min. Oven temperature was held at 50 °C for 5 min, programmed to 160 °C at a rate of 10 °C/min, held at 160 °C for 10 min, programmed to 180 °C at 0.3 °C/min, held to 180 °C for 5 min, and then increased to 240 °C at 20 °C/min, and held at 240 °C for 10 min. The transfer line was held at 240 °C and the mass spectroscopy source at 200 °C. Acquisition was performed in electron ionization mode (70 eV) at 0.4 scan/s, and the mass range used was 10–400 m/z. The identification of the compound was made by using the NIST library , the MS data of literature, and when available, the injection of authentic standards. Fatty acid composition was expressed as g/100 g of total fatty acids of milk and cheese, respectively.Data were analyzed using R software . The effChemical composition and fatty acid (FA) profiles of the herbage selected by cows and of the concentrate offered are reported in p > 0.05). PAST1 showed higher CP (p < 0.01) and lower dry matter , ash (p < 0.05), and EE (p < 0.01) concentrations than PAST2. The differences between the FA profiles were limited to C20:4 c5,c8,c11,c14, the highest in PAST2 (p < 0.05). Considering that the two experimental pastures were grazed at the same phenological stage, these differences may be due to their different botanical composition, as explained by Bovolenta et al. [The supplement level did not influence the chemical composition or the FA profile of the herbage selected by the animals  the chemical composition of milk in terms of fat (3.99 ± 0.043%), protein (3.28 ± 0.008%), and SCS (4.03 ± 0.166 units), and of cheese in terms of DM (66.6 ± 0.206%), fat (53.2 ± 0.364% DM), protein (40.5 ± 0.404 % DM). The milk composition was similar to that reported by Romanzin et al. [p < 0.01) and higher in LOW than in HIGH groups , although its average value was within the range of normality of 15–30 mg/dL proposed by Bendelja et al. [The pasture type and the supplement level did not influence , iso-C14:0 (p < 0.01), iso-C15:0 (p < 0.01), C15:0 (p < 0.01), iso-C16:0 (p < 0.01), ante-iso-C17:0 (p < 0.01), C16:0 (p < 0.05), C20:0 (p < 0.05) were higher in LOW than HIGH milk; instead, the C18:0 (p < 0.01) level was highest in milk from the HIGH group. Moreover, LOW tended to have higher C17:0 (p < 0.10), and iso-C18:0 (p < 0.10) than the HIGH group. Vlaeminck et al. [iso-C14:0, iso-C15:0, C15:0, iso-C16:0 are constituents of the bacterial membrane, and that their variation can reflect variations in the bacterial ruminal population. Indeed, the same authors found that cellulolytic bacteria membranes are particularly rich in iso-FA. Therefore, we supposed that the animals from the experimental groups had different ruminal bacterial populations, with the LOW having higher cellulolytic ruminal bacteria than the HIGH group. This hypothesis reflects the probable highest forage/concentrate ratio of the diet of the LOW group. We had to take into account that linear odd-chain FA, and ante-iso-FA can also derive from mammary gland de-novo synthesis, and that the C17:1/C17:0 is also regulated by the action of the Δ9 desaturase enzyme [p > 0.10) with the only exception being C14:0, which was highest in the LOW group (p < 0.05). Additionally, the C17:1/C17:0 ratio was similar between experimental groups , reinforcing the hypothesis that the differences observed for the saturated FA previously reported were due to a different selection of ruminal bacterial populations.Considering the saturated FA of milk, the concentrations of C14:0 , C18:1 t11 (p < 0.01), CLA (p < 0.05), C18:2 t11,c15 (p < 0.01), C18:3 c9,c12,c15 (p < 0.01), and C20:4 c5,c8,c11,c14 (p < 0.01) were higher in LOW than HIGH milk; instead, the C18:1 c9 (p < 0.01), C18:1 c11 (p < 0.01), and C18:1 c12 (p < 0.01) levels were highest in milk from the HIGH group. Moreover, LOW milk tended to have higher C16:1 c7 (p < 0.10) and the sum of C18: t12/t13/t14 isomers (p < 0.10), and lower C18:1 t6-8-9-10 (p < 0.10) than the HIGH group. It is accepted that C18:3 c9,c12,c15 and C18:2 c9,c12 originated in the feed. The herbage ingested contained more than 40% of the total FA of C18:3 c9,c12,c15; in particular, the level of this FA was 1.17 g/100 g DM and 0.09 g/100 g DM in herbage and concentrate, respectively. Therefore, it is reasonable that the LOW group, fed with a lower level of concentrate, ingested a higher amount of herbage, explaining the differences observed in the concentration of C18:3 c9,c12,c15. Conversely, it seems that the differences of C18:2 c9,c12 in the two diets were not enough to generate variations in this FA in the milk of the experimental groups. Indeed, the level of C18:2 c9,c12 was 0.56 g/100 g DM and 1.50 g/100 g DM in herbage and concentrate, respectively. C18:1 t11 and C18:2 t11,c15 are intermediates of the ruminal biohydrogenation of the dietary unsaturated FA, and, therefore, it can be speculated that the ruminal biohydrogenation was higher in animals receiving a lower level of concentrate in accordance with Khanal et al. [t11, CLA, and C18:3 c9,c12,c15. It is interesting to note that the C18:0 level, a final product of the ruminal biohydrogenation of dietary FA, was higher in the HIGH than LOW group. Taking into account our results, it could be hypothesized that the final step of rumen biohydrogenation was inhibited in the LOW group. Dewanckele et al. [c9 can derive from the desaturation of C18:0, the difference observed in this FA could be due to the different levels of C18:0 between experimental groups.Considering the unsaturated FA of milk, the concentrations of C17:1  was higher, while the level of MUFA was lower (p < 0.01) in LOW than HIGH group. Romanzin et al. [Considering the groups of FA, the level of PUFA , C12:0 (p < 0.05), C16:0 (p < 0.01), C18:1 c14/t16 (p < 0.01), and MUFA (p < 0.01), but lower levels of iso-C14:0 (p < 0.01), iso-C15:0 (p < 0.01), ante-iso-C15:0 (p < 0.05), iso-C16:0 (p < 0.01), ante-iso-C17:0 (p < 0.01), C17:0 (p < 0.01), iso-C18:0 (p < 0.01), C18:1 c12 (p < 0.01), C18:1 t (p < 0.05), and C20:0 (p < 0.01) than the PAST2 group. Despite not being measured, the difference in the herbage intake by animals between PAST1 and PAST2 can be considered very small following the INRA standard [iso-FA, ante-iso-C15:0, and ante-iso-C17:0, we can speculate that the different pasture type was able to influence the ruminal bacteria population. Conversely, in the present study, since only C12:0 of the short chain FAs was different between pastures, the effect of pasture type on mammary de novo synthesis of FA was not evident. Similarly, taking into account that only C18:1 t and C18:1 c12 were different between experimental groups, the effect of pasture type on ruminal biohydrogenation of dietary FA was also not evident. In the literature, the effect of pasture type on milk FA is highly variable. Khiosa-Ard et al. [c9,c12,c15 in milk was influenced by the botanical family of plants ingested by animals, which is not in agreement with the results of the present study. Falchero et al. [Poaceae was correlated positively with MUFA and negatively with C18:2 c9,c12 levels in milk. Conversely, Collomb et al. [Poaceae, reported a significant positive correlation with SFA, a negative significant correlation with PUFA, and no correlation with MUFA. The Alpine pastures are characterized by a high botanical diversity, and different plants can contain different levels of secondary metabolites, such as polyphenol oxidase and tannins, that can influence the metabolism of FA [The pasture type had a more limited effect on FA composition of milk than concentrate levels in animals’ diets. Indeed, PAST1 showed higher levels of C10:1  or type of pasture (p > 0.05). Only the LOW group tended to have higher phytanic acid content than the HIGH group (p < 0.10). Phytanic acid cannot be synthetized by dairy cows and has a dietary origin deriving from the metabolism of chlorophyll in the rumen, while the SRR/RRR diastereomer ratio depends on the microbial flora present in the rumen as a consequence of the animal’s diet [Phytanic acid and its isomers’ SRR/RRR ratio in milk was not affected either by supplement level  showed a level of C12:0 and C16:0 around 1% lower than those produced from cows grazing on Poion alpinae alliance pasture (PAST1 group). Therefore, LOW and PAST2 had a more beneficial FA profile of milk than HIGH and PAST1 groups, respectively. Many studies have reported that SFA, and C12:0, C14:0, and C16:0 in particular, have hypercholesterolemic properties and favor the increase of blood low density lipoprotein ,60. Convpressure . In the The FA composition of experimental cheeses is showed in iso-C15:0 (p < 0.05), iso-C17:0 (p < 0.01), C17:1 (p < 0.01), C18:0 (p < 0.01), C18:1 t11 (p < 0.01), C18:1 c9 (p < 0.01), C18:1 t15 (p < 0.05), and C18:3 c9,c12,c15 (p < 0.01) were higher in LOW than HIGH cheese; instead, the C4:0 (p < 0.01), C8:0 (p < 0.01), C10:0 (p < 0.01), C12:0 (p < 0.01), C13:0 (p < 0.01), C18:1 c12 (p < 0.05), and C20:0 (p < 0.01) levels were highest in cheese from the HIGH group. Coppa et al. [t11, and C18:3 c9,c12,c15 reflected in those of milk, some differences in FA composition in milk and cheese were found. These differences could be due to the lipolysis process that cheese underwent during ripening. From this point of view, one of the most important lipolytic agents is the lipoprotein lipase (LPL) derived from the milk and that is inactivated after pasteurization. This enzyme preferentially acts towards short chain fatty acids, C4-C12 [p < 0.01), and C18:1 t15 (p < 0.01), but lower iso-C14:0 (p < 0.01), iso-C15:0 (p < 0.01), ante-iso-C15:0 (p < 0.05), iso-C16:0 (p < 0.01), C15:0 (p < 0.01), iso-C16:0 (p < 0.01), C17:0 (p < 0.01), C18:1 c12 (p < 0.01), C18:2 CLA (p < 0.05), and C20:0 (p < 0.01) levels than those from the PAST2 group.The concentrations of a et al.  observed, C4-C12 ,63. The , C4-C12  and Cora, C4-C12 . Unlike Considering the categories of FA, it is interesting to note that the cheese-making process and the ripening period reduced the differences between experimental groups, especially considering the supplement level factor. Indeed, the cheese of the HIGH group showed similar SFA, MUFA, and PUFA levels to those of the LOW group. Additionally, the total concentration of these categories of FA was similar between cheese and milk independent of the experimental factor. The average total amounts of SFA, MUFA, and PUFA were 66.9, 27, and 6.1% respectively, which were similar to those reported by Romanzin et al. [p > 0.05).As for milk, phytanic acid level and its SRR/RRR diastereomer ratio were similar between experimental groups (c9,c12,c15, as well as some FA intermediates of rumen biohydrogenation. Conversely, the effect of the two pasture types considered was more limited. From the milk FA profile point of view, increasing the concentrate offered to cows decreased the level of FA beneficial for human health, such as PUFA and C18:3 The differences in PUFA levels in all the experimental cheeses, probably because of the cheese-making process and the ripening period, were reduced. Finally, we can conclude that the increase in the concentrate offered to dairy cows from 1.5 to 3.0 kg/d, which can have a nutritional role, does not markedly influence the level of SFA, MUFA, and PUFA in cheeses produced during summer grazing on different high mountain pastures.


HNF1A-gene single-nucleotide polymorphisms (SNPs) and having early-onset, MODY-like diabetes mellitus in the Turkish population.The molecular basis of the Turkish population with suspected maturity-onset diabetes of the young (MODY) has not been identified. This is the first study to investigate the association between N = 565) who presented to our clinic between 2012 and 2015 with a clinical suspicion of MODY were included in the study. Analysis of HNF1A, HNFB, HNF4A, GCK gene mutations was performed using real-time polymerase chain reaction sequencing. After genetic analysis, diabetics (n = 46) with HNF1A, HNF1B, HNF4A, GCK gene mutations (diagnosed as MODY) and diabetics (n = 30) with HNF1B, HNF4A, GCK gene SNPs were excluded. Patients with early-onset, MODY-like diabetes (n = 486) and non-diabetic controls (n = 263) were included. Genetic analyses for the HNF1A gene p.S487 N (rs2464196), p.A98V (rs1800574) and p.I27L (rs1169288) SNPs were performed using Sanger-based DNA sequencing among the control group.All diabetic patients . p.I27L GT/TT carriers  and p.I27L TT carriers had increased risk of having MODY-like diabetes . Family inheritance of diabetes was significantly more common in patients with the p.I27L TT genotype. The p.I27L SNP was modestly associated with having diabetes after adjusting for body mass index and age .HNF1A gene p.I27L SNP was modestly associated with having early-onset, MODY-like diabetes in the Turkish population. HNF1A gene p.I27L SNP might contribute to age at diabetes diagnosis and family inheritance.The The online version of this article (10.1186/s12902-019-0375-2) contains supplementary material, which is available to authorized users. HNF1A gene has been identified in both monogenic and polygenetic diabetes. Rare mutations of the HNF1A gene cause a monogenic form of diabetes as Type 3 maturity-onset-diabetes of the young (MODY3) [HNF1A gene contributes to the pathogenesis of Type 2 diabetes mellitus (T2DM). HNF1A gene single nucleotide polymorphisms (SNPs) were modestly associated with Type 2 diabetes mellitus (T2DM) and glycemic features in different populations [HNF1A SNPs were associated with impaired insulin secretion [HNF1A gene SNPs  were inconsistently associated with impaired glucose tolerance and having diabetes [HNF1A gene SNPs on developing MODY-like diabetes. This is the first study to investigate the association between HNF1A gene SNPs rs1169288 (encoding HNF1A p.Ile27Leu), rs1800574  and rs2464196 (encoding HNF1A p.Ser486Asn), and having early-onset, MODY-like diabetes in the Turkish population.Hepatocyte nuclear factor 1A (HNF1A) is a transcription factor that has a role in the development and function of pancreas ß-islet cells. In the developmental stage, both endocrine and exocrine cells of the pancreas have HNF1A expression –4. The Hulations –7. HNF1Aecretion , 9. HNF1diabetes . Some yodiabetes . The genn = 263) had diabetes. All patients with diabetes (n = 486) met the criteria for the diagnosis of MODY. Subjects with a clinical suspicion of MODY [n = 565) in order to diagnose MODY. After genetic analysis, patients with diabetes (n = 46) who had HNF1A, HNF1B, HNF4A, GCK gene mutations were diagnosed as having MODY3, MODY5, MODY1, and MODY2 respectively. Thirty patients with diabetes had HNF1B, HNF4A, had GCK gene SNPs. Patients with diabetes with MODY and HNF1B, HNF4A, and GCK SNPs were excluded from the study. Finally, subjects without HNF1A, HNF1B, HNF4A, and GCK gene mutations and HNF1B, HNF4A, and GCK SNPs (n = 486) and non-diabetic healthy controls (n = 263) were included this study.In our study, none of the control subjects . BMI ≥ 30 kg/m2 was diagnosed as obesity. T2DM was diagnosed when plasma fasting glucose concentrations were > 125 mg/dL, casual or postprandial glucose levels were > 200 mg/dL or in the presence of current treatment with a hypoglycemic agent, according to the American Diabetes Association criteria [Fasting glucose, postprandial glucose, creatinine, HbA1c, triglycerides (TG), cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), C-peptide, high-sensitivity (hs-CRP), anti-glutamic acid decarboxylase (GAD) antibody, anti-insulin antibody, anti-islet antibody, and urinary microalbuminuria concentrations were measured. Age and symptoms at onset of diabetes, diabetes treatment, and parental history of diabetes  were recorded from all patients with diabetes. Body mass index (BMI) was calculated as weight (kg) / height  was similar between the diabetics and controls (p > 0.05). The mean age of the controls was 49.18 ± 3.38 years. The mean age at onset of diabetes was 24.08 ± 4.82 years. The mean C-peptide concentration of the patients with diabetes was 2.47 ± 1.79 nmol/L. Fasting glucose, HbA1c, TG, cholesterol, LDL-C concentrations were higher among the diabetics compared with the controls , and p.S487 rs2464196 were not consistent with the HWE . p.A98V SNPs were similar between the diabetics and controls in the dominant model and recessive model . The p.A98V TT genotype was higher in diabetics in the codominant model compared with the controls . HNF1A gene p.I27L TT genotype was increased in diabetes  compared with the controls in the codominant model. p.I27L GT/TT carriers had increased 1.68 odds of having diabetes  in the dominant model. p.I27L TT carriers had 1.56-fold increased odds of having diabetes  in the recessive model. Clinical and biochemical characteristics did not differ between patients with diabetes with p.I27L, p.S487 N, and p.A98V SNPs and diabetics without the SNPs (p > 0.05). Onset of diabetes was 26.17 ± 7.4 years in p.S487 N, 25.58 ± 2.7 years in p.A98V, and 24.57 ± 5.2 years in p.I27L (p > 0.05). Parent diabetes (mother or father) was higher in the p.I27L TT genotype compared with the GG genotype . Diabetics with p.I27L TT genotype had higher triglyceride concentrations compared with diabetics with the GG genotype (p = 0.041) .The percentage of women (51.5% vs. 58.2%) and BMI value  and having late-onset T2DM in the European population [HNF1A gene SNPs and having late-onset T2DM [p.A98V SNPs were included [p.I27L and p.A98V SNPs were associated with having late-onset T2DM [p.I27L GT/TT carriers had 1.68-fold increased odds of having diabetes, and p.I27L TT carriers had 1.5 6-fold increased odds of having diabetes in our study. Only the p.I27L variant was modestly associated with having diabetes and this relationship continued after adjusting BMI and age. There was no association between p.S487 N and p.A98V SNPs and early-onset T2DM. Similar to our report, a modest association was shown between p.I27L, p.S487 N, and p.A98V and having T2DM in the European population [p.I27L was associated with having T2DM in non-obese French [p.I27L was associated with having T2DM [HNF1A gene p.I27L was associated with having late-onset T2DM in Brazilian [p.I27L was associated with having late-onset T2DM in overweight Scandinavian subjects aged over 60 years, and p.A98V was reported to decrease in vivo glucose-responsive to insulin secretion [p.I27L has a modest role in β-cell dysfunction [p.A98V and having T2DM [p.A98V to be associated with decreased insulin secretion in healthy individuals [HNF1A gene p.A98V was associated with having early-onset T2DM in Scandinavian [HNF1A p.A98V was associated with having late-onset T2DM in Finnish but not in Chinese individuals [p.A98V TT genotype was higher compared with the GG genotype in diabetics, nevertheless, with no association.In this study, we suggest that polygenic T2DM may show differences in age-related and family inheritance transmission for an associated monogenic form of diabetes. This is the first study to show the effect of the p.I27L genotype on modifying age at diagnosis in the Turkish population. We excluded monogenic diabetes modifier genes, which often include mutations, because we aimed to examine the influence of variations on polygenic diabetes. In our study, subjects with diabetes were non-obese and the onset of diabetes was early. A previous study showed that non-obese patients with early-onset diabetes were more susceptible to β-cell dysfunction as compared with old and obese individuals . A modesset T2DM , but a rincluded . Similarset T2DM . p.I27L pulation . In agree French  and Finne French . A Chineing T2DM . HNF1A grazilian  and Westrazilian  overweigrazilian . In linefunction  and in ifunction . Consisting T2DM , 4. A Daividuals . HNF1A gdinavian  and Asiaividuals . Our stu.I27L + p.S487 N carriers [p.I27L + p.S487 N ± p.A98V carriers, as compared with HNF1A mutation (14 years). Locke et al. reported that each p.I27L allele was associated with a 1.6-year decrease in age at diagnosis in patients with HNF1A-MODY [HNF1A gene SNPs. Similar to our report, paternal diabetes was higher in HNF1A gene SNP carriers [p.I27L homozygous TT carriers, suggesting a probability of significant familial transmission.Early-onset diabetes (19 years) was observed in Chinese pcarriers . Yorifujcarriers . A GermaF1A-MODY . Our stucarriers . This stHNF1A locus p.I27L is localized in the dimerization domain, p.S487 N is localized in the transactivation domain, and the p.A98V is localized in the DNA-binding domain [HNF1A gene p.I27L, p.A98V, and p.S487 N variants reduce transcriptional activities of genes that have a role in glucose metabolism [p.I27L + p.A98V variations decreased transactivation activity on GLUT2 in HeLa cells more than p.I27L alone and p.A98V alone [HNF1A controls ß-cell function by regulating target genes such as glucose transporter 2 (GLUT2), HNF 4A, collectrin, liver pyruvate kinase, and hepatocyte growth factor activator. HNF1A activity dysfunction causes a reduction β-cell mass and induces onset of diabetes [HNF1A variation can be explained by environmental factors together with epigenetic factors [The g domain , 28. HNF8V alone , 25, 31. factors , 31.p.I27L and p.A98V were consistent with the HWE whereas p.S487 was not consistent with HWE. HNF1A gene p.I27L and p.A98V haplotypes were within LD. These are the limitations of this study.This study had a case-control design and small sample size. HNF1A gene age at diagnosis that shows an effect of genetic variation on diabetes phenotype. The HNF1A variant p.I27L was associated with having early-onset, MODY-like diabetes in the Turkish population. Enlightening the role of HNF1A in β-cells would be helpful in understanding the molecular mechanism of both T2DM and MODY and would guide new therapeutic approaches.We report a genetic modifier of the Additional file 1:Genotyping and Statistical analysis. (DOCX 14 kb)


A total of 205 animals from four Egyptian livestock species; cattle (n = 18), buffaloes (n = 12), sheep (n = 150) and goats (n = 25) were used in this study to detect polymorphism and perform comparative analysis for IGFBP-3 gene using DNA sequencing and (PCR–RFLP).HaeIII restriction enzyme yielded a single restriction pattern for goats, while for cattle, 3 genotypes were identified; (AA), (AB), and (BB). Moreover, for buffalo one genotype (AA) only was found with HaeIII and TaqI restriction enzymes, separately. Also, the digestion profile for goats with HaeIII revealed one pattern only. Nucleotide sequencing of the amplified fragments of IGFBP-3 gene in sheep, cattle, buffalo, and goat was submitted to the NCBI GenBank . The nucleotide sequencing analysis indicated similarity percentages in IGFBP-3 gene fragments of 88.54, 89.63 and 95.06% between “sheep and cattle”, “sheep and buffalo”, and “cattle and buffalo”, respectively.The amplified fragments were found to be of length 654 bp in sheep, 651 bp in cattle and 655 bp in buffalo. For Falahy goats, PCR was performed to amplify a 316 bp fragment from exon 2 of the IGFBP-3 gene. The digestion of 654 bp with  There are inherent limitations in genome analysis of farm animal species in Egypt. Not enough genetic investigations on these animals are available, whether at the level of genes, QTL, or whole genome. Therefore, we tried through this modest attempt to open a window for studying the genome of these species, starting with some genes that have a major effect on some economic traits such as IGFBP-3. To the best of our knowledge, no studies are, yet, available on the comparisons in IGFBP-3 gene among domestic sheep, goat, cattle and buffalo species of Egypt , 2. AddiInsulin-like growth factor binding proteins (IGFBPs) belongs to a family of at least six homologous proteins that bind IGFs and modulate many of their important biological actions . It is rEgyptian breeds from different species require the determination and characterization of several candidate genes responsible for the alteration of the economic traits behaviour. The objective of this study was to detect polymorphism in IGFBP-3 gene in Egyptian sheep, Falahy goats, Egyptian cattle and El-Beheiry buffaloes using a comparative sequence analysis (DNA sequencing) and PCR–RFLP techniques on samples of livestock species.Samples from four Egyptian farm animal species, namely cattle , buffaloes , sheep  and goats  were obtained from four different geographical regions at the northern coast of Egypt, namely; Baltim farm —Al Burlos —Alexandria University ‘experimental station’  and Matrouh farm . Blood samples of 5 ml each were collected from the jugular vein, using venojects, treated with 0.5 ml of 2.7% EDTA  as an anticoagulant, kept in an icebox and transferred immediately to the lab. Sampled animals were apparently unrelated and their general characteristics correspond to the respective breeds conformation.Genomic DNA was extracted from blood samples with QIAGEN  according to the manufacturer’s instructions. The isolated DNAs were separated by electrophoresis on 0.8% agarose  in 0.5 X TBE buffer prepared according to Sambrook et al.  and contFor the sampled Egyptian sheep, cattle, and buffaloes, a region of IGFBP-3 gene spanning over a part of exon 2, complete intron 2, exon 3 and a part of intron 3 was amplified using primer AASN-P1; (F: 5-CCAAGC GTG AGA CAG AAT AC-3),(R:5-AGG AGG GAT AGG AGC AAG AT-3) . PCR forThe amplification was performed using , 10 p.mol of each primer and 80–100 ng of genomic DNA were processed under the following amplification conditions: 94 °C/5 min, followed by 35 cycles of 94 °C/1 min, 60 °C/1 min, 72 °C/1 min and a final extension step at 72 °C/2 min for (AASN-P1) primer. As for (AASN-P2) primer, the conditions were 94 °C/5 min, followed by 34 cycles of 94 °C/1 min, 63 °C/1 min, 72 °C/1 min and a final extension step at 72 °C/2 min. The amplification was carried out using a DNA Thermo-cycler Gene Amp 6700 .http://www.ncbi.nlm.nih.gov). The resulted sequences were analyzed using MEGA 6 v.4, Finch T.V 1.01, and Blast 2.0 software to detect Single Nucleotide Polymorphism (SNPs) between sequences. The sequences were deposited in GenBank . Analysis of translated protein of IGFBP-3 gene sequences of tested animals were generated by ExPASy program (http://web.expasy.org/translate).Automated DNA sequence analysis was carried out on both strands by the DNA sequencing service lab of the Korean Research Institute of Bioscience and Biotechnology with an ABI Prism 3100 apparatus. Database similarity searches were performed with the NCBI/BLAST/blast network service at the National Center for Biotechnology Information (NCBI) . Defining restrictions sites before digestion with restriction enzymes was achieved by the NEB cutter program (http://www.labtools.us/nebcutter-v2-0) [2O, 2 μl 10X digestion buffer, 5–10 units restriction enzyme (5 unit/1 μl) in addition to 10 μl amplified DNA. All reactions were incubated at 37 °C for HaeIII and at 65 °C for TaqI for 16 h. Twenty microliter of each reaction were separated by electrophoresis on 3% agarose gel and visualized by UV trans-illuminator and gel documentation system .The RFLP was used to detect genotyping differences between and within sheep groups, cattle, buffalo, and goat using the PCR of target genes. The PCR amplicons of the IGFBP-3 gene were digested with er-v2-0) . The RFLHaeIII  at the base number 300 of IGFBP-3 gene sequence indicating also, an absence of polymorphism at this site.The present results agree with those of Kumar et al.  who study et al.  reportedHaeIII revealed three genotypes in some local cattle;(AA), (CC), and (AC) with frequencies nearly 22%, 22%, and 56%, respectively.For Egyptian cattle, three genotypes were identified: 1) Lanes 1 and 2; homozygous (BB) genotype with 8 restricted fragments at 199, 164, 154, 56, 36, 18, 16 and 8 bp, (2) Lanes 3 and 4; heterozygous (AB) genotype with 5 restricted fragments at 199, 194, 164, 56 and 36 bp and (3) Lanes 5 and 6; homozygous (AA) genotype with 8 restricted fragments at 194, 164, 159, 56, 36, 18, 16 and 8 bp  with restriction fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp  of the amplified IGFBP-3 gene fragments.Additional file https://blast.ncbi.nlm.nih.gov/Blast) indicated that the similarity percentages of IGFBP-3 gene fragment between (sheep and cattle was 88.54%), between , while between    and the comparison of amino acids obtained by MEGA-6 VERSION 4 are in  - Digestion pattern of PCR amplification of IGFBP-3 gene from the genomic DNA of tested buffalo breeds with Taq-I revealing a single homozygous genotype and a non-polymorphic as two fragments of sizes 415 and 240 bp were observed, M; 50 bp DNA ladder. (B) - A 655 bp sequence of IGFBP-3 gene of Egyptian buffalo (NCBI accession no. MG738674) in this study with restriction sites for the HaeIII restriction enzyme, thus all screened buffaloes showed only one genotype (AA) with restriction fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp. Figure S2. Digestion pattern of PCR amplification of the IGFBP-3 gene from the genomic DNA of tested goat breeds, the digested with HaeIII revealed one pattern only for three DNA fragments sized 263, 58 and 8 bp, the restriction fragment with size 8 bp was not seen on the gel, M; 50 bp DNA ladder. Figure S3. The diagrammatic representation of exon–intron regions of animals tested and restriction enzymes sites (HaeIII and TaqI) of amplified IGFBP-3 gene fragment in sheep, cattle, buffalo, and goat. Figure S4. (A) - A 654 bp sequence of the IGFBP-3 gene for Egyptian sheep (NCBI accession no. MG738671.1). (B)- A 651 bp sequence of IGFBP-3 gene for Egyptian Cattle (NCBI accession no. MG738673.1). (C) - A 655 bp sequence of IGFBP-3 gene for Egyptian buffalo (NCBI accession no. MG738674.1). (D) - A 316 bp sequence of IGFBP-3 gene for Egyptian goat (NCBI accession no. MG738672.1) in the current study. Figure S5. Nucleotide sequence comparison of amplified IGFBP-3 gene of sheep, cattle and buffalo using (MEGA-6) Molecular Evolutionary Genetics Analysis, VERSION 4 (http://en.bio-soft.net/tree/MEGA.html). Figure S6. Comparative analysis of protein sequence of IGFBP-3 gene of sheep, cattle and buffalo using ExPASy program (http://web.expasy.org/translate) and the comparison of amino acid using (MEGA-6), VERSION 4 (http://en.bio-soft.net/tree/MEGA.html).