Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment

We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD₅₀ of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.     

Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner

Active transport of NaCl across thick ascending limb (TAL) epithelium is accomplished by Na(+),K(+),2Cl(-) cotransporter (NKCC2). The activity of NKCC2 is determined by vasopressin (AVP) or intracellular chloride concentration and includes its amino-terminal phosphorylation. Co-expressed Tamm-Horsfall protein (THP) has been proposed to interact with NKCC2. We hypothesized that THP modulates NKCC2 activity in TAL. THP-deficient mice (THP(-/-)) showed an increased abundance of intracellular NKCC2 located in subapical vesicles (+47% compared with wild type (WT) mice), whereas base-line phosphorylation of NKCC2 was significantly decreased (-49% compared with WT mice), suggesting reduced activity of the transporter in the absence of THP. Cultured TAL cells with low endogenous THP levels and low base-line phosphorylation of NKCC2 displayed sharp increases in NKCC2 phosphorylation (+38%) along with a significant change of intracellular chloride concentration upon transfection with THP. In NKCC2-expressing frog oocytes, co-injection with THP cRNA significantly enhanced the activation of NKCC2 under low chloride hypotonic stress (+112% versus +235%). Short term (30 min) stimulation of the vasopressin V2 receptor pathway by V2 receptor agonist (deamino-cis-D-Arg vasopressin) resulted in enhanced NKCC2 phosphorylation in WT mice and cultured TAL cells transfected with THP, whereas in the absence of THP, NKCC2 phosphorylation upon deamino-cis-D-Arg vasopressin was blunted in both systems. Attenuated effects of furosemide along with functional and structural adaptation of the distal convoluted tubule in THP(-/-) mice supported the notion that NaCl reabsorption was impaired in TAL lacking THP. In summary, these results are compatible with a permissive role for THP in the modulation of NKCC2-dependent TAL salt reabsorptive function.     

The Combined Effects of Bacterial Symbionts and Aging on Life History Traits in the Pea Aphid,Acyrthosiphon pisum

While many endosymbionts have beneficial effects on hosts under specific ecological conditions, there can also be associated costs. In order to maximize their own fitness, hosts must facilitate symbiont persistence while preventing symbiont exploitation of resources, which may require tight regulation of symbiont populations. As a host ages, the ability to invest in such mechanisms may lessen or be traded off with demands of other life history traits, such as survival and reproduction. Using the pea aphid, Acyrthosiphon pisum, we measured survival, lifetime fecundity, and immune cell counts (hemocytes, a measure of immune capacity) in the presence of facultative secondary symbionts. Additionally, we quantified the densities of the obligate primary bacterial symbiont, Buchnera aphidicola, and secondary symbionts across the host''s lifetime. We found life history costs to harboring some secondary symbiont species. Secondary symbiont populations were found to increase with host age, while Buchnera populations exhibited a more complicated pattern. Immune cell counts peaked at the midreproductive stage before declining in the oldest aphids. The combined effects of immunosenescence and symbiont population growth may have important consequences for symbiont transmission and maintenance within a host population.     

Patterns in attachment sites of the parasite Hydrachna guanajuatensis Cook, 1980 (Acari: Hydrachnidiae) on aquatic heteropterans (Nepomorpha) from the Tecocomulco Lake,Mexico

The water mite Hydrachna guanajuatensis Cook, 1980 Cook, D. (1980), ‘Studies on Neotropical Water Mites’ Memoirs of the American Entomological Institute, 31, 1–645. [Google Scholar] is recorded as an ectoparasite of Ranatra sp. (Nepidae), Notonecta sp., Buenoa sp. (Notonectidae), Krizousacorixa femorata (Guérin, 1857), Trichocorixella mexicana (Hungerford, 1927), and Hesperocorixa laevigata (Uhler, 1893) (Corixidae). The host body region, where the larvae and protonymph attach, depends significantly on the heteropteran species: χ² = 3969.43, p < 0.05 and χ² = 1893.6, p < 0.05, respectively. We performed a cross tabulation with a correspondence analysis and found three associations for the larvae: 1) dorsal side of abdomen underneath the wings of the two species of Notonectidae, 2) head, wings, and hind legs of two of the species of Corixidae (T. mexicana and K. femorata), and 3) thorax and fore and middle legs of the single species of Nepidae. These different patterns are discussed according to morphological and ecological characteristics of the hosts. This is the first study that deals with the spatial distribution patterns within the same species of ectoparasite.     

Environmental drivers of ectomycorrhizal communities in Europe's temperate oak forests

Ectomycorrhizal fungi are major ecological players in temperate forests, but they are rarely used in measures of forest condition because large‐scale, high‐resolution, standardized and replicated belowground data are scarce. We carried out an analysis of ectomycorrhizas at 22 intensively monitored long‐term oak plots, across nine European countries, covering complex natural and anthropogenic environmental gradients. We found that at large scales, mycorrhizal richness and evenness declined with decreasing soil pH and root density, and with increasing atmospheric nitrogen deposition. Shifts in mycorrhizas with different functional traits were detected; mycorrhizas with structures specialized for long‐distance transport related differently to most environmental variables than those without. The dominant oak‐specialist Lactarius quietus, with limited soil exploration abilities, responds positively to increasing nitrogen inputs and decreasing pH. In contrast, Tricholoma, Cortinarius and Piloderma species, with medium‐distance soil exploration abilities, show a consistently negative response. We also determined nitrogen critical loads for moderate (9.5–13.5 kg N/ha/year) and drastic (17 kg N/ha/year) changes in belowground mycorrhizal root communities in temperate oak forests. Overall, we generated the first baseline data for ectomycorrhizal fungi in the oak forests sampled, identified nitrogen pollution as one of their major drivers at large scales and revealed fungi that individually and/or in combination with others can be used as belowground indicators of environmental characteristics.     

Atrial Fibrillation and Beta Thalassemia Major: The Predictive Role of the 12-lead Electrocardiogram Analysis

Background

Paroxysmal atrial tachyarrhythmias frequently occur in beta-thalassemia major (β-TM) patients.The aim of our study was to investigate the role of maximum P-wave duration (P max) and dispersion (PD), calculated trough a new manually performed measurement with the use of computer software from all 12-ECG-leads,as predictors of atrial-fibrillation (AF) in β-TM patients with conserved systolic or diastolic cardiac function during a twelve-months follow-up.

Materials and Methods

50 β-TM-patients (age38.4±10.1; 38M) and 50-healthy subjects used as controls, matched for age and gender, were studied for the occurrence of atrial arrhythmias during a 1-year follow-up, through ECG-Holter-monitoring performed every three months. The β-TM-patients were divided into two groups according to number and complexity of premature-supraventricular-complexes at the Holter-Monitoring (Group1: <30/h and no repetitive forms, n:35; Group2: >30/h or couplets, or run of supraventricular tachycardia and AF, n:15).

Results

Compared to the healthy control-group, β-TM patients presented increased P-max (107.5± 21.2 vs 92.1±11ms, P=0.03) and PD-values (41.2±13 vs 25.1±5 ms,P=0.03). In the β-TM population, the Group2 showed a statistically significant increase in PD (42.8±8.6 vs 33.2±6.5ms, P<0.001) and P-max (118.1±8.7 vs 103.1±7.5ms, P<0.001) compared to the Group1. Seven β-TM patients who showed paroxysmal AF during this study had significantly increased P-max and PD than the other patients of the Group2. Moreover, P-max (OR:2.01; CI:1.12-3.59; P=0.01) and PD (OR=2.06;CI:1.17-3.64;P=0.01) demonstrated a statistically significant association with the occurrence of paroxysmal AF,P min was not associated with AF-risk (OR=0.99; CI:0.25-3.40; P=0.9) in β-TM-patients. A cut-off value of 111ms for P-max had a sensitivity of 80% and a specificity of 87%, a cut-off value of 35.5ms for PD had a sensitivity of 90% and a specificity of 85% in identifying β-TM patients at risk for AF.

Conclusion

Our results indicate that P-max and PD are useful electrocardiographic markers for identifying the β-TM-high-risk patients for AF onset, even when the cardiac function is conserved.     

Matrix‐assisted peptide synthesis on nanoparticles

We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix‐assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.