COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina

Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.     

Antioxidant responses of cortex neurons to iron loading

Brain cells have a highly active oxidative metabolism, yet they contain only low to moderate superoxide dismutase and catalase activities. Thus, their antioxidant defenses rely mainly on cellular reduced glutathione levels. In this work, in cortical neurons we characterized viability and changes in reduced and oxidized glutathione levels in response to a protocol of iron accumulation. We found that massive death occurred after 2 days in culture with 10 microM Fe. Surviving cells developed an adaptative response that included increased synthesis of GSH and the maintenance of a glutathione-based reduction potential. These results highlight the fundamental role of glutathione homeostasis in the antioxidant response and provide novel insights into the adaptative mechanisms of neurons subjected to progressive iron loads.     

Costs and Epidemiological Changes of Chronic Diseases: Implications and Challenges for Health Systems

Background

The need to integrate economic and epidemiological aspects in the clinical perspective leads to a proposal for the analysis of health disparities and to an evaluation of the health services and of the new challenges which are now being faced by health system reforms in middle income countries.

Objective

To identify the epidemiological changes, the demand for health services and economic burden from chronic diseases (diabetes and hypertension) in a middle income county.

Methods

We conducted longitudinal analyses of costs and epidemiological changes for diabetes and hypertension in the Mexican health system. The study population included both the insured and uninsured populations. The cost-evaluation method was used, based on the instrumentation and consensus techniques. To estimate the epidemiological changes and financial consequences for 2014–2016, six models were constructed according to the Box-Jenkins technique, using confidence intervals of 95%, and the Box-Pierce test.

Results

Regarding epidemiological changes expected in both diseases for 2014 vs. 2016, an increase is expected, although results predict a greater increase for diabetes, 8–12% in all three studied institutions, (p < .05). Indeed, in the case of diabetes, the increase was 41469 cases for uninsured population (SSA) and 65737 for the insured population (IMSS and ISSSTE). On hypertension cases the increase was 38109 for uninsured vs 62895 for insured. Costs in US$ ranged from $699 to $748 for annual case management per patient in the case of diabetes, and from $485 to $622 in patients with hypertension. Comparing financial consequences of health services required by insured and uninsured populations, the greater increase (23%) will be for the insured population (p < .05). The financial requirements of both diseases will amount to 19.5% of the total budget for the uninsured and 12.5% for the insured population.

Conclusions

If the risk factors and the different health care models remain as they currently are, the economic impact of expected epidemiological changes on the social security system will be particularly strong. Another relevant challenge is the appearance of internal competition in the use and allocation of financial resources with programs for other chronic and infectious diseases.     

Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure

Adaptation to high and low copper intake in mammals depends on the cellular control of influx, efflux and storage mechanisms of cellular copper concentrations. In the present study, we used an intestinal cell line (Caco-2), grown in bicameral chambers to study the effect of equilibrium loading with copper. We analyzed (64)Cu uptake from the apical surface, intracellular metal (Cu, Zn, Fe) content, (64)Cu transport into the basal chamber, and total copper, zinc and iron in the basal chamber. We found that the (64)Cu uptake is saturable, shows a linear response phase up to 1.5 microM reaching a plateau at 4-6 microM extracellular Cu. Intracellular copper increased 21.6-fold, from 1.5 to 32.4 mM (at 0.2-20.2 microM extracellular copper respectively). The time course for (64)Cu uptake and transport was linear when the cells were incubated with different copper concentrations. Uptake increased 10-fold when intracellular copper concentration was raised. Fluxes were lowest at 1.5 mM and highest at 32.4 mM Cu intracellular copper (2.03 and 20. 98 pmole (64)Cu insert(-1) h(-1), respectively). The apical-to-basolateral copper transfer rate was lower at 32.4 mM as compared to 1.5 mM intracellular copper (0.55-1.95 pmole (64)Cu insert(-1) h(-1), respectively). The total copper in the basal chamber increased 4.2-fold (from 3.04 to 12.85 pmole Cu insert(-1) h(-1)) when the intracellular copper concentration was raised. If cells are preincubated in a low copper medium most of the newly incorporated copper (64%) is transferred to the basolateral compartment. In contrast, under preloading with high copper concentration, only 4% of the fresh copper is transferred to the basal chamber; however, the intracellular copper contribution to this chamber increases by 4.2-fold. Thus, the process results in an increase in both storage and intracellular-to-basolateral flux of copper. In summary, our results indicate that copper fluxes from apical-to-cell and apical-to-basolateral domains are affected by intracellular copper concentration suggesting that mechanisms of copper transport involved in cellular adaptation to low and high copper exposure are different.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).     

Rapid immunodetection of Escherichia coli

A filtration flow-through design was used to develop the rapid immunodetection of Escherichia coli. Polyclonal anti-E. coli IgG was conjugated to small, 0.8 Blue latex beads. Cells were mixed with conjugated beads in the presence of anti-E. coli monoclonal IgM. The suspension was then filtered through a 5 nitrocellulose membrane. The cell-containing complexes were effectively collected on the filter, forming a blue spot. The method produced reliable detection of E. coli at a concentration of 10⁵ cells ml^–1, which is a current benchmark figure for urinary tract infection (UTI) diagnosis.     

Determinants of Performance of Health Systems Concerning Maternal and Child Health: A Global Approach

Aims

To assess the association of social determinants on the performance of health systems around the world.

Methods

A transnational ecological study was conducted with an observation level focused on the country. In order to research on the strength of the association between the annual maternal and child mortality in 154 countries and social determinants: corruption, democratization, income inequality and cultural fragmentation, we used a mixed linear regression model for repeated measures with random intercepts and a conglomerate-based geographical analysis, between 2000 and 2010.

Results

Health determinants with a significant association on child mortality(<1year): higher access to water (βa Quartile 4(Q4) vs Quartile 1(Q1) = -6,14; 95%CI: -11,63 to -0,73), sanitation systems, (Q4 vs Q1 = -25,58; 95%CI: -31,91 to -19,25), % measles vaccination coverage (Q4 vs Q1 = -7.35; 95%CI: -10,18 to -4,52), % of births attended by a healthcare professional (Q4 vs Q1 = -7,91; 95%CI: -11,36 to -4,52) and a % of the total health expenditure (Q3 vs Q1 = -2,85; 95%CI: -4,93 to -0,7). Ethnic fragmentation (Q4 vs Q1 = 9,93; 95%CI: -0.03 to 19.89) had a marginal effect. For child mortality<5 years, an association was found for these variables and democratization (not free vs free = 11,23; 95%CI: -0,82 to 23,29), out-of-pocket expenditure (Q1 vs Q4 = 17,71; 95%CI: 5,86 to 29,56). For MMR (Maternal mortality ratio), % of access to water for all the quartiles, % of access to sanitation systems, (Q3 vs Q1 = -171,15; 95%CI: -281,29 to -61), birth attention by a healthcare professional (Q4 vs Q1 = -231,23; 95%CI: -349,32 to -113,15), and having corrupt government (Q3 vs Q1 = 83,05; 95%CI: 33,10 to 133).

Conclusions

Improving access to water and sanitation systems, decreasing corruption in the health sector must become priorities in health systems. The ethno-linguistic cultural fragmentation and the detriment of democracy turn out to be two factors related to health results.     

Quantifying the reduction in potential health risks by determining the sensitivity of poliovirus type 1 chat strain and rotavirus SA-11 to electron beam irradiation of iceberg lettuce and spinach

Fresh produce, such as lettuce and spinach, serves as a route of food-borne illnesses. The U.S. FDA has approved the use of ionizing irradiation up to 4 kGy as a pathogen kill step for fresh-cut lettuce and spinach. The focus of this study was to determine the inactivation of poliovirus and rotavirus on lettuce and spinach when exposed to various doses of high-energy electron beam (E-beam) irradiation and to calculate the theoretical reduction in infection risks that can be achieved under different contamination scenarios and E-beam dose applications. The D(10) value (dose required to reduce virus titers by 90%) (standard error) of rotavirus on spinach and lettuce was 1.29 (± 0.64) kGy and 1.03 (± 0.05) kGy, respectively. The D(10) value (standard error) of poliovirus on spinach and lettuce was 2.35 (± 0.20) kGy and 2.32 (± 0.08) kGy, respectively. Risk assessment of data showed that if a serving (～14 g) of lettuce was contaminated with 10 PFU/g of poliovirus, E-beam irradiation at 3 kGy will reduce the risk of infection from >2 in 10 persons to approximately 6 in 100 persons. Similarly, if a serving size (～0.8 g) of spinach is contaminated with 10 PFU/g of rotavirus, E-beam irradiation at 3 kGy will reduce infection risks from >3 in 10 persons to approximately 5 in 100 persons. The results highlight the value of employing E-beam irradiation to reduce public health risks but also the critical importance of adhering to good agricultural practices that limit enteric virus contamination at the farm and in packing houses.     

Identification and characterization of the biotechnological potential of a wild strain of Paraconiothyrium sp

The isolation and characterization of fungal strains from poorly described taxa allows undercover attributes of their basic biology useful for biotechnology. Here, a wild fungal strain (CMU‐196) from recently described Paraconiothyrium genus was analyzed. CMU‐196 was identified as Paraconiothyrium brasiliense by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS). CMU‐196 metabolized 57 out of 95 substrates of the Biolog FF microplates. Efficient assimilation of dextrins and glycogen indicates that CMU‐196 is a good producer of amylolytic enzymes. It showed a remarkably assimilation of α‐d ‐lactose, substrate described as inducer of cellulolytic activity but poorly assimilated by several fungi. Metabolically active mycelium of the strain decolorized broth supplemented with direct blue 71, Chicago sky blue and remazol brilliant blue R dyes. The former two dyes were also well removed from broth by mycelium inactivated by autoclaving. Both mycelia had low efficiency for removing fuchsin acid from broth and for decolorizing wastewater from the paper industry. CMU‐196 strain showed extracellular laccase activity when potato dextrose broth was supplemented with Cu⁺², reaching a maximum activity of 46.8 (±0.33) U L^?1. Studied strain antagonized phytopathogenic Colletotrichum spp. fungi and Phytophthora spp. oomycetes in vitro, but is less effective towards Fusarium spp. fungi. CMU‐196 antagonism includes overgrowing the mycelia of phytopathogens and growth inhibition, probably by hydrosoluble extracellular metabolites. The biotechnological potential of strain CMU‐196 here described warrants further studies to have a more detailed knowledge of the mechanisms associated with its metabolic versatility, capacity for environmental detoxification, extracellular laccase production, and antagonism against phytopathogens. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:846–857, 2018     

The Ras/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediates cholinergic regulation of keratinocyte directional migration

The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell re-orientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by high-performance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of alpha7 nicotinic and M(1) muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from alpha7 and M(1) through the MEK1/ERK was an up-regulated expression of alpha(2) and alpha(3) integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, alpha7 works together with M(1) to orient a keratinocyte toward direction of its future migration. Both alpha7 and M(1) apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge.