Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs

Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain.     

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site.     

Application of next-generation sequencing methods for microbial monitoring of anaerobic digestion of lignocellulosic biomass

The anaerobic digestion of lignocellulosic wastes is considered an efficient method for managing the world’s energy shortages and resolving contemporary environmental problems. However, the recalcitrance of lignocellulosic biomass represents a barrier to maximizing biogas production. The purpose of this review is to examine the extent to which sequencing methods can be employed to monitor such biofuel conversion processes. From a microbial perspective, we present a detailed insight into anaerobic digesters that utilize lignocellulosic biomass and discuss some benefits and disadvantages associated with the microbial sequencing techniques that are typically applied. We further evaluate the extent to which a hybrid approach incorporating a variation of existing methods can be utilized to develop a more in-depth understanding of microbial communities. It is hoped that this deeper knowledge will enhance the reliability and extent of research findings with the end objective of improving the stability of anaerobic digesters that manage lignocellulosic biomass.     

Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas

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Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.     

Structure,Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABA_B receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment.     

Genetic diversity of the common bacterial blight pathogen of bean, <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis>, in Iran revealed by rep-PCR and PCR–RFLP analyses

Xanthomonad-like bacteria that are associated with common bacterial blight of bean in Iran were identified on the basis of their colonial morphology, biochemical and serological properties, presence of a specific DNA fragment using PCR primers and pathogenicity on bean. Xanthomonas axonopodis pv. phaseoli (Xap) strains were further characterized using rep-PCR and restriction fragment length polymorphism (RFLP). RFLP profiles generated by the restriction endonucleases RsaI, TaqI, HaeIII and Sau96I and rep-PCR analysis revealed that Iranian strains were relatively genetically homogenous. The similarity coefficients among the strains ranged from 0.87 to 1. The genetic diversity coefficients among strains from three infected provinces, Isfahan, Markazi and Lorestan, were 0.019, 0.072 and 0.033, respectively. The low overall level of polymorphism within Xap isolates collected from the three Iranian infected regions could suggest that few initial inoculum introductions might have distributed among these different bean-growing areas in Iran.     

Efficacy of a Cladosporium sp. fungus against Helicoverpa armigera (Lepidoptera: Noctuidae), other insect pests and beneficial insects of cotton

A strain of the fungus Cladosporium sp. (RM16) from an egg of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) was assessed as a potential biocontrol agent for this pest. Pathogenicity of the fungus was tested against H. armigera eggs and larvae, cotton aphids (Aphis gossypii Glover; Homoptera: Aphididae), and silverleaf whitefly type B (Bemisia tabaci Gennadius; Hemiptera: Aleyrodidae). The pathogenicity of the fungus to the predatory red and blue beetles (Dicranolaius bellulus Guérin-Méneville; Coleoptera: Melyridae), transverse ladybird beetles (Coccinella transversalis Fabricius; Coleoptera: Coccinellidae), green lacewings (Mallada signatus Schneider; Neuroptera: Chrysopidae) and damsel bugs (Nabis kinbergii Reuter; Hemiptera: Nabidae), was also assessed in the laboratory. Fungus treatment resulted in failure to hatch of up to 64% of H. armigera eggs (compared with 11% in the controls) and mortality of 54% of first instar H. armigera larvae (compared with 5% in the controls). In contrast, it was not pathogenic to later instar H. armigera larvae. Cladosporium RM16 was also efficacious against the sap-sucking insect pests of cotton that were tested. No significant harmful effect of the fungus was found on any of the four beneficial predatory insects assessed in this study. Cladosporium RM16 has the potential as biological control agent to support integrated pest management in cotton farming systems, although this needs intensive study.