Assessing potentiation of the (α4)3(ß2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI.

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two ß2 subunits ((α4)3(ß2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh)-binding sites located in the two α4:ß2 intersubunit interfaces and a low-affinity "noncanonical" ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:ß2 interfaces), TC-2559 (which binds at the α4:ß2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:ß2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(ß2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod-Wyman-Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:ß2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(ß2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.

Examining the Effects of (α4)3(ß2)2 Nicotinic Acetylcholine Receptor-Selective Positive Allosteric Modulator on Acute Thermal Nociception in Rats.

Neuronal nicotinic acetylcholine receptor (nAChR)-based therapeutics are sought as a potential alternative strategy to opioids for pain management. In this study, we examine the antinociceptive effects of 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrazol-4-yl)isoxazole (CMPI), a novel positive allosteric modulator (PAM), with preferential selectivity to the low agonist sensitivity (α4)3(ß2)2 nAChR and desformylflustrabromine (dFBr), a PAM for α4-containing nAChRs. We used hot plate and tail flick tests to measure the effect of dFBr and CMPI on the latency to acute thermal nociceptive responses in rats. Intraperitoneal injection of dFBr, but not CMPI, dose-dependently increased latency in the hot plate test. In the tail flick test, the effect achieved at the highest dFBr or CMPI dose tested was only <20% of the maximum possible effects reported for nicotine and other nicotinic agonists. Moreover, the coadministration of dFBr did not enhance the antinociceptive effect of a low dose of nicotine. Our results show that the direct acute effect of dFBr is superior to that for CMPI, indicating that selectivity to (α4)3(ß2)2 nAChR is not advantageous in alleviating responses to acute thermal nociceptive stimulus. However, further studies are necessary to test the suitability of (α4)3(ß2)2 nAChR-selective PAMs in chronic pain models.

Unraveling amino acid residues critical for allosteric potentiation of (α4)3(ß2)2-type nicotinic acetylcholine receptor responses.

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(ß2)2 but not (α4)2(ß2)3 nAChR isoforms. This selective potentiation underlines that the α4:α4 interface, which is present only in the (α4)3(ß2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the α4:α4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues α4Gly-41, α4Lys-64, and α4Thr-66 were critical for (α4)3(ß2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at α4His-116, a known determinant of NS9283 and of agonist binding at the α4:α4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at α4Gln-124 and α4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the α4:α4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (α4)3(ß2)2 nAChR.

Impact of microplastic pollution on coastal ecosystems using comprehensive beach quality indices.

Previous studies of Microplastics (Mps) pollution focused on abundance, effect on organisms, and origins. Mps could also be indicators to evaluate pollution level. Beach Quality Indices (BQIs) are useful in understanding Mps pollution level. This study is to assess magnitude, impact and quality of beaches using BQIs, by determining abundance, shape, and size of Mps in beach sediments, which is the first effort in China. Three BQIs, i.e. Microplastic Pollution Index (MPPI), Environmental Status Index (ESI), Coefficient of Microplastic Impact (CMPI), were employed involving Sector Analysis Approach. All beaches had "very high" abundance by MPPI, were classified "bad" by ESI, and fell in "red" sector using Sector Analysis Approach by intergradation of MPPI and ESI. The impact of fiber morphology was "extreme" based on CMPI. The average abundance was 664±80 Mps/kg. Fibers occupied >97 % of Mps, with 31 % of black Mps. A model was proposed to determine Mps origins.

Microplastics pollution along the central Atlantic coastline of Morocco.

This baseline study describes the microplastic (MPs) problem on seven beaches located on Agadir, central Atlantic coast of Morocco. Microplastics abundances (densities) ranged from 7680 MPs/kg to 34,200 MPs/kg above other world beaches. The following shapes were found: fibers, fragments, films, and pellets. Fibers were the dominant typology with a 73%. Polyethylene (PE) was the predominant polymer in all beaches with an average percentage of 59%, followed by Polypropylene (PP - 18%), Polystyrene (PS - 9%), Polyvinyl chloride (PVC - 8%), and Ethylene-vinyl acetate (EVA - 6%). Microplastics abundances presented in this work suggest continuous inputs of plastics of all sizes and types. Also, all observed shapes highlight a combination of sources (primary and secondary). Central Atlantic Moroccan beaches are currently affected by extreme values of MPs that demand urgent interventions to restore environmental quality.

The transcription factor complex CmAP3-CmPI-CmUIF1 modulates carotenoid metabolism by directly regulating carotenogenic gene CmCCD4a-2 in chrysanthemum.

Carotenoids are one of the most important pigments for the coloring in many plants, fruits and flowers. Recently, significant progress has been made in carotenoid metabolism. However, the specific understanding on transcriptional regulation controlling the expression of carotenoid metabolic genes remains extremely limited. Anemone-type chrysanthemum, as a special group of chrysanthemum cultivars, contain elongated disc florets in capitulum, which usually appear in different colors compared with the ray florets since accumulating distinct content of carotenoids. In this study, the carotenoid composition and content of the ray and disc florets of an anemone-type chrysanthemum cultivar 'Dong Li Fen Gui' were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and the key structural gene CmCCD4a-2, of which differential expression resulted in the distinct content of carotenoids accumulated in these two types of florets, was identified. Then the promoter sequence of CmCCD4a-2 was used as bait to screen a chrysanthemum flower cDNA library and two transcription factors, CmAP3 and CmUIF1 were identified. Y2H, BiFC and Y3H experiments demonstrated that these two TFs were connected by CmPI to form CmAP3-CmPI-CmUIF1 TF complex. This TF complex regulated carotenoid metabolism through activating the expression of CmCCD4a-2 directly. Furthermore, a large number of target genes regulated directly by the CmAP3-CmPI-CmUIF1 TF complex, including carotenoid biosynthetic genes, flavonoid biosynthetic genes and flower development-related genes, were identified by DNA-affinity purification sequencing (DAP-seq), which indicated that the CmAP3-CmPI-CmUIF1 TF complex might participate in multiple processes. These findings expand our knowledge for the transcriptional regulation of carotenoid metabolism in plants and will be helpful to manipulating carotenoid accumulation in chrysanthemum.

Massive hematemesis and melena stool: A case report of unusual manifestation of Cow's milk protein intolerance.

Cow's milk protein intolerance (CMPI) is a common condition that causes gastrointestinal bleeding in the first year of life. It is the most common cause of chronic blood loss and anemia; however, severe massive hematemesis is an uncommon condition. Herein, we present a case of severe massive hematemesis with melena stool in a six-month-old boy with cow's milk protein intolerance. In this case, we described management used in poor developing countries.

Microplastics pollution on Colombian Central Caribbean beaches.

The growing literature on microplastics (MPs) in coastal and marine environs reflects the seriousness of this pollutant category. Diverse litter studies on Colombia's Central Caribbean Coast have not presented detailed study of MPs' typology, magnitude or distribution. This baseline study presents for first time the MPs problem on 23 beaches in 75 km coastal reach between Punta Roca and Galerazamba, on the central Colombian Caribbean Coast. The Microplastics Pollution Index (MPPI) and Coefficient of Microplastic Impact (CMPI) were developed and applied along with the Environmental Status Index (ESI) and their integration through sector analysis, and mapping using Agglomerative Hierarchical Clustering and Multidimensional Scaling, and Principal Components Analysis. Microplastics abundances (densities) ranged from 2457 to 557 MPs/kg, similar to other global beaches and bays. The MPs Index for the 23 beaches classified 8 beaches as "Moderate," 10 beaches as "High" and 5 as "Very High." Microplastic fibers were the dominant typology at 83% of the combined beaches total (ranging from Moderate to Very High for individual beaches).

How can ocean warming at the NW Iberian Peninsula affect mussel aquaculture?

Understanding and forecasting future consequences of climate change in mussel aquaculture industry require the assessment of changes in physical parameters which may affect mussel growth. The FLOW module of Delft3D model forced with climatic data was validated and calibrated for the Rías Baixas (NW Iberian Peninsula), one of the areas with the highest mussel production in the world. This model was used to perform historical (1999-2018) and future (2080-2099) projections. Temperature and stratification water conditions were compared in order to determine at what extent climate change can affect mussel production. Thermal stress will increase in a non-homogeneous throughout the water column and the comfort level of mussels will be reduced by more than 60% in the upper layers and more than 30% in deep layers in most of the mussel raft polygons. Water column stratification will increase ~ 5-10 cycles h-1 in most of the polygons reducing the vertical exchange of nutrients and oxygen. Hereby changes in water temperature and stratification at the end of the century will not be favorable for mussel growth.

The impact of climate change on the geographical distribution of habitat-forming macroalgae in the Rías Baixas.

In the current scenario of climate change characterized by a generalized warming, many species are facing local extinctions in areas with conditions near their thermal tolerance threshold. At present, the southern limit of the geographical distribution of several habitat-forming algae of cold-temperate affinities is located in the Northwest Iberian Peninsula, and the Rías Baixas may be acting as contemporary refugia at the range edge. Therefore, it is necessary to analyze future changes induced by ocean warming in this area that may induce changes in macroalgae populations. The Delft3D-Flow model forced with climatic data was used to calculate July-August sea surface temperature (SST) for the present (1999-2018) and for the far future (2080-2099). Mean daily SST was used to develop and calibrate a mechanistic geographical distribution model based on the thermal survival threshold of two intertidal habitat-forming macroalgae, namely Himanthalia elongata (L.) S.F.Gray and Bifurcaria bifurcata R. Ross. Results show that H. elongata will become extinct in the Rías Baixas by the end of the century, while B. bifurcata will persist and may occupy potential free space left by the decline in H. elongata.

Quantitation of free and total N-acetylcysteine amide and its metabolite N-acetylcysteine in human plasma using derivatization and electrospray LC-MS/MS.

Studies of N-acetylcysteine amide (NACA) in nonclinical models have demonstrated various antioxidant, anti-apoptotic, anti-inflammatory and neuroprotective effects, and it is currently being developed as a treatment for retinitis pigmentosa. Sensitive LC-MS/MS methods were developed and validated to quantitate reduced and total NACA and its major metabolite, N-acetylcysteine (NAC), in human plasma to support clinical studies involving NACA. To trap and stabilize reduced NACA and NAC at the time of collection, whole blood was immediately treated with 2-chloro-1-methylpyridinium iodide (CMPI) to convert free thiols to 1-methylpyridinyl thioether derivatives. Plasma was harvested and frozen until samples were assayed using protein precipitation and an LC-MS/MS separation based on hydrophilic-interaction chromatography (HILIC). To process NACA and NAC present as disulfides, an intermediate portion of the extract was further subjected to reduction with tris(2-carboxyethyl) phosphine; the released thiols were then reacted with CMPI, extracted, and analyzed as before, to measure total thiols. The method for NACA and NAC, whether free/reduced or total, covered a range from 50 ng/mL to 50 µg/mL in human plasma and required a single 25 µL plasma sample. Up to 180 samples could be assayed in a single session. The inter-run mean bias and precision (%CV) were within ±5% for the free thiol method and within ±8.5% for the total thiol method. Benchtop, freeze/thaw, and long-term stability were evaluated and acceptable. The NAC/NACA method applied to a clinical study demonstrated incurred sample reproducibility of 95.5% for NAC and 99.1% for NACA.

Hyaluronic acid auto-crosslinked polymer (ACP): Reaction monitoring, process investigation and hyaluronidase stability.

Hyaluronic Acid (HA) is a non-sulphated glycosaminoglycan that, despite its high molecular weight, is soluble in water and is not resistant to enzymatic degradation, the latter of which hinders its wider application as a biomedical material. Auto-crosslinked polymer (ACP) gels of HA are fully biocompatible hydrogels that exhibit improved viscoelastic properties and prolonged in vivo residence times compared to the native polymer. Crosslinking is achieved through a base-catalysed reaction consisting of the activation of HA carboxyl groups by 2-chloro-1-methylpyridinium iodide (CMPI) and subsequent nucleophilic acyl substitution by the hydroxyl groups of HA in organic solvent. In this study, a number of ACP hydrogels have been obtained via reactions using varying ratios of CMPI to HA. The crosslinking reaction was monitored by rheological measurements in organic solvents during CMPI addition to the reaction mixture. The ACP intermediates, powders and hydrogels were characterized, helping to elucidate the crosslinking process. A two-step mechanism was proposed to explain the observed trends in viscosity and particle size. Syntheses were carried out by varying the reaction temperature, respectively at 0 °C, 25 °C and 45 °C in N-Methyl-2-Pyrrolidone (NMP), as well as the solvent respectively in NMP, DMSO and DMF at 25 °C. Interestingly, varying these parameters did not substantially affect the degree of crosslinking but likely did influence the intra/inter-molecular crosslinking ratio and, therefore, the viscoelastic properties. A wide range of crosslinking densities was confirmed through ESEM analysis. Finally, a comparative hyaluronidase degradation assay revealed that the ACPs exhibited a higher resistance toward enzymatic cleavage at low elastic modulus compared to other more chemically resistant, crosslinked HAs. These observations demonstrated the importance of crosslinking density of matrix structures on substrate availability.