Desmoglein versus non-desmoglein signaling in pemphigus acantholysis: characterization of novel signaling pathways downstream of pemphigus vulgaris antigens

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces "desmosomal" signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by approximately 45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by approximately 50% and TUNEL positivity by approximately 25%. The p38 MAPK inhibitor PD169316 inhibited these effects by approximately 15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked approximately 50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.     

Calculation of the free energy and cooperativity of protein folding

Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design.     

RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site

Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.     

Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host

Despite increasing evidence of behavioural manipulation of their vectors by pathogens, the underlying mechanisms causing infected vectors to act in ways that benefit pathogen transmission remain enigmatic in most cases. Here, 2-D DIGE coupled with MS were employed to analyse and compare the head proteome of mosquitoes (Anopheles gambiae sensu stricto (Giles)) infected with the malarial parasite (Plasmodium berghei) with that of uninfected mosquitoes. This approach detected altered levels of 12 protein spots in the head of mosquitoes infected with sporozoites. These proteins were subsequently identified using MS and functionally classified as belonging to metabolic, synaptic, molecular chaperone, signalling, and cytoskeletal groups. Our results indicate an altered energy metabolism in the head of sporozoite-infected mosquitoes. Some of the up-/down-regulated proteins identified, such as synapse-associated protein, 14-3-3 protein and calmodulin, have previously been shown to play critical roles in the CNS of both invertebrates and vertebrates. Furthermore, a heat shock response (HSP 20) and a variation of cytoarchitecture (tropomyosins) have been shown. Discovery of these proteins sheds light on potential molecular mechanisms that underlie behavioural modifications and offers new insights into the study of intimate interactions between Plasmodium and its Anopheles vector.     

Canopy Light Gradient Perception by Cytokinin

We have recently identified cytokinin as an important xylem-carried signal involved in the photosynthetic acclimation of plants to light gradients in dense canopies. Lower leaves become shaded in a dense canopy and consequently have reduced transpiration rates. our measurements have shown that this results in a reduced delivery of cytokinins carried in the transpiration stream to shaded leaves, as compared to light-exposed leaves. Cytokinins are involved in the regulation of photosynthetic acclimation to the light gradient by stimulating the expression of photosynthetic enzymes in light-exposed leaves. In shaded leaves, the low delivery rate of cytokinin leads to reduced photosynthetic capacity and ultimately senescence. We show evidence for this role of cytokinin, as part of a complex of signaling pathways where other regulatory mechanisms are also involved. A model is presented depicting the regulation of photosynthetic acclimation by cytokinin delivery to leaves dependent on the irradiance they receive.Key Words: canopy light gradient, transpiration, photosynthetic acclimation, cytokinin, nitrate, systemic signaling     

Spatially resolved abundances of antibiotic resistance genes and intI1 in wastewater treatment biofilms

Attached growth bioprocesses that use biofilms to remove organic matter or nutrients from wastewater are known to harbor antibiotic resistance genes (ARGs). Biofilms in these processes are spatially heterogeneous, but little is known about depth stratification of ARGs in complex, mixed culture biofilms. To address this knowledge gap, we used an experimental approach combining cryosectioning and quantitative polymerase chain reaction to quantify the spatial distribution of three ARGs (sul1, ermB, and qnrS) and the class 1 integron-integrase gene intI1 in biofilms from a lab-scale rotating annular reactor fed with synthetic wastewater. We also used high throughput 16S ribosomal RNA (rRNA) gene sequencing to characterize community structure with depth in biofilms. The ARG sul1 and the integron-integrase gene intI1 were found in higher abundances in upper layers of biofilm near the fluid-biofilm interface than in lower layers and exhibited significant correlations between the distance from substratum and gene abundances. The genes ermB and qnrS were present in comparatively low relative abundances. Microbial community structure varied significantly by date of sampling and distance from the substratum. These findings highlight the genetic and taxonomic heterogeneity with distance from substratum in wastewater treatment biofilms and show that sul1 and intI1 are particularly abundant near fluid-biofilm interfaces where cells are most likely to detach and flow into downstream portions of treatment systems and can ultimately be released into the environment through effluent.