Structural basis for cooperative interactions of substituted 2-aminopyrimidines with the acetylcholine binding protein

The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are pentameric oligomers in which binding sites for nicotinic agonists and competitive antagonists are found at selected subunit interfaces. The nAChR spontaneously exists in multiple conformations associated with its activation and desensitization steps, and conformations are selectively stabilized by binding of agonists and antagonists. In the nAChR, agonist binding and the associated conformational changes accompanying activation and desensitization are cooperative. AChBP, which lacks the transmembrane spanning and cytoplasmic domains, serves as a homology model of the extracellular domain of the nAChRs. We identified unique cooperative binding behavior of a number of 4,6-disubstituted 2-aminopyrimidines to Lymnaea AChBP, with different molecular variants exhibiting positive, n_H > 1.0, and negative cooperativity, n_H < 1.0. Therefore, for a distinctive set of ligands, the extracellular domain of a nAChR surrogate suffices to accommodate cooperative interactions. X-ray crystal structures of AChBP complexes with examples of each allowed the identification of structural features in the ligands that confer differences in cooperative behavior. Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epibatidine. An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, noncooperative, nicotinic ligands. Positively and negatively cooperative ligands exhibited unique features in the detailed binding determinants and poses of the complexes.Nicotinic acetylcholine receptors (nAChRs) function as allosteric pentamers of identical or homologous transmembrane spanning subunits. Ligand binding at two or more of the five intersubunit sites, located radially in the extracellular domain, drives a conformational change that results in the opening of a centrosymmetric transmembrane channel, internally constructed among the five subunits (SI Appendix, Fig. S2A) (1–4). Up to five potential agonist-competitive antagonist sites on the pentamer are found at the outer perimeter of the subunit interfaces. Amino acid side-chain determinants on both subunit interfaces dictate selectivity among the many subtypes of nAChRs. The interconversion between resting, active, and desensitized states occurs in the absence of ligands, and partial occupation of the binding sites suffices for agonist activation of the receptor and its antagonism (5–7). Cooperativity of agonist association and its coupling to channel gating likely play important roles in the dynamics of nicotinic responses and in sharpening the concentration and temporal windows for activation.As revealed in functional studies, most nAChRs are hetero-oligomeric, where the sites of ligand occupation are not identical (1–4). This arrangement arises when a common α-subunit pairs with one or more nonidentical subunit partners, termed non–α-subunits (7, 8). Nonidentity of the subunit interface complementary to the α-subunit may also give rise to heterogeneity in binding constants typically seen for antagonists and mask partially the degree of agonist cooperativity. An exception to this is the α7-neuronal nAChR composed of five identical subunits and exhibiting a high degree of cooperativity for agonist activation (9). Recently, sequence alignments identified genes coding for pentameric ligand-gated ion channels in prokaryotes led to the resolution of the first structure by X-ray crystallography on 3D crystals of a pentameric receptor protein from Erminia chrysanthemi (ELIC) (10) and Gloeobacter violaceus (GLIC) (11, 12) and provided high-resolution structures of the two end point states of the cooperative gating mechanism in the same pentameric ligand-gated ion channel (GLIC) (13). Recently, the first structure of a eukaryotic member of the family, the anionic glutamate receptor from Caenorhabditis elegans (GluCl), was solved at atomic resolution (14), revealing remarkable identity of 3D structure with GLIC.The acetylcholine binding protein (AChBP) was characterized from mollusks (15–17) and consists of only a homologous extracellular domain of the nAChR. Assembled as a homomeric pentamer, AChBP exhibits a similar profile of ligand selectivity toward the classical nicotinic agonists and antagonists of quaternary amine, tertiary and secondary amine (alkaloid), imine, and peptide origin that bind nicotinic receptors (18–25). If looked at solely on the basis of ligand-binding capacities, AChBP could be considered as a distinct subtype of nAChR. Although its homomeric composition and ligand selectivity best resemble the α7-subtype of nAChR, when the concentration dependence of ligand occupation has been examined, no evidence of cooperativity emerged (21). Accordingly the cooperative behavior for both activation and desensitization of receptors, seen for the classical nicotinic agonists with nAChRs, might arise from a cooperative torsional motion driven by the transmembrane spanning domain of the receptor (26).We demonstrate here a set of ligands that bind to the AChBP in a cooperative fashion, whereby binding to a single subunit affects the binding energy at identical interfaces in the pentamer. Hence, interactions within the extracellular domain of this family of homologous pentameric proteins establish a circumferential linkage between subunit interfaces which results in cooperative behavior.     

Land,irrigation water,greenhouse gas,and reactive nitrogen burdens of meat,eggs, and dairy production in the United States

Livestock production impacts air and water quality, ocean health, and greenhouse gas (GHG) emissions on regional to global scales and it is the largest use of land globally. Quantifying the environmental impacts of the various livestock categories, mostly arising from feed production, is thus a grand challenge of sustainability science. Here, we quantify land, irrigation water, and reactive nitrogen (Nr) impacts due to feed production, and recast published full life cycle GHG emission estimates, for each of the major animal-based categories in the US diet. Our calculations reveal that the environmental costs per consumed calorie of dairy, poultry, pork, and eggs are mutually comparable (to within a factor of 2), but strikingly lower than the impacts of beef. Beef production requires 28, 11, 5, and 6 times more land, irrigation water, GHG, and Nr, respectively, than the average of the other livestock categories. Preliminary analysis of three staple plant foods shows two- to sixfold lower land, GHG, and Nr requirements than those of the nonbeef animal-derived calories, whereas irrigation requirements are comparable. Our analysis is based on the best data currently available, but follow-up studies are necessary to improve parameter estimates and fill remaining knowledge gaps. Data imperfections notwithstanding, the key conclusion—that beef production demands about 1 order of magnitude more resources than alternative livestock categories—is robust under existing uncertainties. The study thus elucidates the multiple environmental benefits of potential, easy-to-implement dietary changes, and highlights the uniquely high resource demands of beef.Appreciation of the environmental costs of food production has grown steadily in recent years (e.g., refs. 1–3), often emphasizing the disproportionate role of livestock (4–12). Although potentially societally important, to date the impacts of this research on environmental policies (7, 13, 14) and individual dietary choices have been modest. Although pioneering early environmental burden estimates have tended to address wide food classes (notably the animal-based portion of the diet; e.g., refs. 9 and 15), most policy objectives and individual dietary choices are item specific.For example, a person may consider beef and chicken mutually interchangeable on dietary or culinary grounds. However, even if an individual estimate of the environmental cost of one item exists, it is often not accompanied by a directly comparable study of the considered alternative. Even in the unlikely event that both estimates are available, they are unlikely to consider the costs in terms of more than one metric, and often rely on disparate methodologies. Therefore, environmentally motivated dietary choices and farm policies stand to benefit from more finely resolved environutritional information. Although early work yielded a short list of item-specific environmental cost estimates (16), those estimates were often based on meager data, and addressed a single environmental metric (typically energy), thus requiring expansion, updating, and further analysis to enhance statistical robustness (8).Current work in the rapidly burgeoning field of diet and agricultural sustainability falls mostly into two complementary approaches. The first is bottom–up, applying rigorous life cycle assessment (LCA) methods to food production chains (17–22). Whereas early LCAs focused primarily on greenhouse gas (GHG) emissions (23–26), or in some cases GHGs and energy use (5, 27), more recent LCAs often simultaneously address several additional key metrics (17, 19–21, 28, 29), notably land, water, and reactive nitrogen (Nr, nitrogen fertilizer) use. Some studies also include emissions of such undesirable gases (in addition to GHGs) as smog precursors or malodors (30, 31), or adverse contributions to stream turbidity or erosional topsoil loss (e.g., refs. 32–34). This bottom–up approach is extremely important, and is poised to eventually merge with the top–down national efforts described in the next paragraph. This merger is not imminent, however, because the bottom–up approach considers one or at most a handful of farms at a time. Because of wide differences due to geography (35), year-to-year fluctuations (36), and agrotechnological practice (17, 37), numerous LCAs are required before robust national statistics emerge. Eventually, when a large and diverse LCA sample is at hand, the picture at the national level will emerge. Currently, however, the results from an LCA conducted in Iowa, for example, are unlikely to represent Vermont or Colorado. Given the current volume and scope of LCA research, and the complexity and variability of the problem, LCAs are still too few and too local to adequately sample the multifaceted, diverse US food system, and thus to collectively become nationally scalable.The second agricultural sustainability research thrust, into which this study broadly falls, is a top–down analysis of national (10, 16, 38) or global (8, 39–41) production statistics. The top–down approach we follow here is conceptually straightforward, as described schematically in Fig. 1. The environmental needs (land, irrigation water, etc.) of feed production are collected and distributed among the feed-consuming animal categories. This is termed the partitioning step, and is based on information about the number of animals raised or slaughtered mass in each category, as well as the characteristic feed ration in each category. The burdens attributed to each category are divided by the caloric or protein mass output of that animal category, yielding the final result, the environmental burden per consumed unit (e.g., agricultural land needed per ingested kilocalorie of poultry). This method is mainly appealing because it (i) circumvents the variability issues raised above by using national or global aggregations; and (ii) it is based on relatively solid data. For the United States in particular, US Department of Agriculture (USDA) data tend to be temporally consistent, nearly all-inclusive (e.g., records of the main crops are based on close to 100% of the production), and are reported after some (albeit modest) quality control. The key challenge with this approach is obtaining defensible numerical values and uncertainty ranges for the tens if not hundreds of parameters needed in the calculations, many of which are poorly constrained by available data. Such parameters include, for example, the average feed required per animal per day or per kilogram of weight gain, or the relative fraction of pasture in beef and dairy diets. The values vary as a function of, at least, season, geographical location, and agrotechnology used. One research effort, focused on a single location, is unlikely to yield definitive results. Significant progress in both approaches is primarily realized through the tenacious and painstaking amassing of many independent analyses over time; analyses from which robust, meaningful statistics can be derived. Because of the challenges associated with each of the research thrusts discussed above, quantitatively robust, multimetric estimates that are comparable across different categories and represent the average national environmental burdens have yet to be devised. Although estimates of total national energy use and GHG emissions by agriculture do exist (e.g., refs. 4, 5, 42, and 43), they require further statistical evaluation. The costs in terms of land, irrigation water, and Nr are even less certain.Open in a separate windowFig. 1.A simplified schematic representation of the information flow in calculating environmental burdens per consumed calorie or gram of protein. Feed supply and requirements (blue boxes at top) previously yielded (38) the fraction of each feed class consumed by each animal category; e.g., pork requires 23 ± 9% of concentrated feed. Combined with the environmental burdens (green boxes at left; land, irrigation water, and nitrogen fertilizer for each of the three feed classes), these fractions yield the burdens attributed to each animal category. Finally, dividing those overall environmental burdens attributed to each of the five livestock categories by the number of calories (or grams of protein) nationally consumed by humans in the United States, we reach the final result of this paper (yellow box at bottom). Most input data (left and top boxes) is known with relative accuracy based on USDA data, whereas environmental burdens of pasture and average feed requirements are less certain.Applying a top–down, uniform methodology throughout, here we present estimates of land, irrigation water, GHG, and Nr requirements of each of the five main animal-based categories in the US diet—dairy, beef, poultry, pork, and eggs—jointly providing 96% of the US animal-based calories. We do not analyze fish for two reasons. First, during the period 2000–2013, fish contributed ≈14 kcal per person per day, ≈0.5% of the total and 2% of the animal-based energy (750 kcal per person per day) in the mean American diet (44). In addition, data addressing feed use by fisheries and aquaculture are very limited and incomplete (relative to the five categories considered). We do not claim to cover all important environmental impacts of livestock production. Rather, we focus on key metrics that can be reliably defined and quantified at the national level with currently available data.     

Clinical and epidemiological features of Guillain‐Barré syndrome in the Western Balkans

The aim of this study was to define features of Guillain‐Barré syndrome in a large cohort of patients from three Western Balkans countries. Data from adult Guillain‐Barré syndrome (GBS) cases from 2009 to 2013 were retrospectively obtained from all tertiary health care centers. During the 5‐year period, 327 new cases of GBS were identified with a male to female ratio of 1.7 : 1. The most common GBS variants were demyelinating (65%) and axonal (12%). At nadir 45% of patients were chair‐bound, confined to bed, or required assisted ventilation, while 5% died. The crude incidence of GBS in Serbia and Montenegro was 0.93 per 100,000 population, and age‐adjusted incidence according to the world standard population was 0.86 per 100,000. Incidence was particularly high in 50‐ to 80‐year‐old men. Statistically significant seasonal variations of GBS were not observed. This study of patients with GBS in the Western Balkans allows us to prepare the health system better and to improve the management of patients. This study also opens opportunities for international collaboration and for taking part in the multinational studies on GBS.     

Tricho-rhino-phalangeal syndrome in a 13-year-old girl with chronic renal failure and severe growth retardation

Introduction: The tricho-rhino-phalangeal syndrome type III (TRPS III) is a rare autosomal dominantly inherited condition. The main clinical features are sparse and slow-growing hair and nails, a pear-shaped nose with a bulbous tip, elongated and flat philtrum, thin upper lip, cone-shaped epiphyses of the phalanges, and short stature. All patients have a point mutation in the TRPS1 gene. Case report: In this paper, we present a 13-year-old female with the typical clinical features of TRPS III, extreme growth retardation, severe deformities of both proximal radii resulting in limited extension of the elbows, and chronic renal failure (CRF) in addition. Molecular diagnostics revealed a missense mutation in exon 6 of TRPS1 that she inherited from her father who is also affected with TRPS III, but does not have CRF. In the index patient, the CRF was found to be due to bilateral renal hypodysplasia (RHD). Conclusion: Beside the renal dysplasia, the girl had severe deformities of the proximal radii – findings which have not been reported so far in TRPS III.     

Concept Analysis of the Patient Reported Outcomes Measurement Information System (PROMIS®) and the International Classification of Functioning,Disability and Health (ICF)

Purpose

The Patient Reported Outcomes Measurement Information System (PROMIS ^® ) is a US National Institutes of Health initiative that has produced self-report outcome measures, using a framework of physical, mental, and social health defined by the World Health Organization in 1948 (WHO, in Preamble to the Constitution of the World Health Organization as adopted by the International Health Conference, New York, 1948). The World Health Organization’s International Classification of Functioning, Disability and Health (ICF) is a comprehensive classification system of health and health-related domains that was put forward in 2001. The purpose of this report is to compare and contrast PROMIS and ICF conceptual frameworks to support mapping of PROMIS instruments to the ICF classification system .

Methods

We assessed the objectives and the classification schema of the PROMIS and ICF frameworks, followed by content analysis to determine whether PROMIS domain and sub-domain level health concepts can be linked to the ICF classification.

Results

Both PROMIS and ICF are relevant to all individuals, irrespective of the presence of health conditions, person characteristics, or environmental factors in which persons live. PROMIS measures are intended to assess a person’s experiences of his or her health, functional status, and well-being in multiple domains across physical, mental, and social dimensions. The ICF comprehensively describes human functioning from a biological, individual, and social perspective. The ICF supports classification of health and health-related states such as functioning, but is not a specific measure or assessment of health, per se. PROMIS domains and sub-domain concepts can be meaningfully mapped to ICF concepts.

Conclusions

Theoretical and conceptual similarities support the use of PROMIS instruments to operationalize self-reported measurement for many body function, activity and participation ICF concepts, as well as several environmental factor concepts. Differences observed in PROMIS and ICF conceptual frameworks provide a stimulus for future research and development.     

Palladium(II) Complexes with N‐Heteroaromatic Bidentate Hydrazone Ligands: The Effect of the Chelate Ring Size and Lipophilicity on in vitro Cytotoxic Activity

Novel Pd(II) complex with N‐heteroaromatic Schiff base ligand, derived from 8‐quinolinecarboxaldehyde (q8a) and ethyl hydrazinoacetate (haOEt), was synthesized and characterized by analytical and spectroscopy methods. The structure of novel complex, as well as structures of its quinoline and pyridine analogues, was optimized by density functional theory calculations, and theoretical data show good agreement with experimental results. A cytotoxic action of the complexes was evaluated on cultures of human promyelocytic leukemia (HL‐60), human glioma (U251), rat glioma (C6), and mouse fibrosarcoma (L929) cell lines. Among investigated compounds, only complexes with quinoline‐based ligands reduce the cell numbers in a dose‐dependent manner in investigated cell lines. The observed cytotoxic effect of two isomeric quinoline‐based complexes is predominantly mediated through the induction of apoptotic cell death in HL‐60 cell line. The cytotoxicity of most efficient novel Pd(II) complex is comparable to the activity of cisplatin, in all cell lines investigated.     

Photodynamic antibacterial effect of graphene quantum dots

Synthesis of new antibacterial agents is becoming increasingly important in light of the emerging antibiotic resistance. In the present study we report that electrochemically produced graphene quantum dots (GQD), a new class of carbon nanoparticles, generate reactive oxygen species when photoexcited (470 nm, 1 W), and kill two strains of pathogenic bacteria, methicillin-resistant Staphylococcus aureus and Escherichia coli. Bacterial killing was demonstrated by the reduction in number of bacterial colonies in a standard plate count method, the increase in propidium iodide uptake confirming the cell membrane damage, as well as by morphological defects visualized by atomic force microscopy. The induction of oxidative stress in bacteria exposed to photoexcited GQD was confirmed by staining with a redox-sensitive fluorochrome dihydrorhodamine 123. Neither GQD nor light exposure alone were able to cause oxidative stress and reduce the viability of bacteria. Importantly, mouse spleen cells were markedly less sensitive in the same experimental conditions, thus indicating a fairly selective antibacterial photodynamic action of GQD.     

Epizootiological survey of small mammals as Leptospira spp. reservoirs in Eastern Croatia

In this survey we investigated a population of small mammals in Eastern Croatia in order to determine Leptospira carriage rates and identify circulating serovars. Out of 67 trapped animals, 20 (29.9%) isolates were obtained. Identification of isolates using microscopic agglutination test, pulsed field gel electrophoresis and multi locus sequence typing revealed that 10 (50.0%) isolates belong to serogroup Pomona, serovar Mozdok, 6 (30.0%) isolates to serogroup Australis, serovar Jalna, 2 (10.0%) isolates to serogroup Sejroe, serovar Saxkoebing, and 1 (5.0%) isolate to serogroup Grippotyphosa, serovar Grippotyphosa. One isolate from serogroup Bataviae was unable to be identified to the serovar level. Amplification of a 331-bp region of the locus LA0322 using real-time polymerase chain reaction determined that 12 (60.0%) isolates belong to L. kirschneri, 6 (30.0%) isolates to L. interrogans, and 2 (10.0%) isolates to L. borgpetersenii. Leptospira carriage rate was high (29.9%), which corresponds to a high incidence of human and domestic animal leptospirosis in Eastern Croatia. Furthermore, 90.0% of the isolates belong to serogroups Pomona, Australis and Sejroe which are also the most prevalent serogroups in humans in this area. These findings suggest that small mammals might be an important source of Leptospira spp. infection in Eastern Croatia.