Kv7 channels can function without constitutive calmodulin tethering

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.     

Intensity of the vaccine-elicited immune response determines tumor clearance.

Tumor Ag-specific vaccines used for cancer immunotherapy can generate specific CD8 responses detectable in PBMCs and in tumor-infiltrating lymphocytes. However, human studies have shown that detection of a systemic vaccine-induced response does not necessarily correlate with the occasional instances of tumor rejection. Because this discrepancy might partially be attributable to the genetic heterogeneity of human cancers, as well as to the immunosuppressive effects of previous treatments, we turned to a mouse model in which these variables could be controlled to determine whether a relationship exists between the strength of vaccine-induced immune responses and tumor rejection. We challenged mice with the beta-galactosidase (beta-gal)-expressing tumor cells, C25.F6, vaccinated them with beta-gal-carrying viral vectors, and used quantitative RT-PCR to measure the vaccine-induced immune response of splenocytes directly ex vivo. We found that the strength of the response increased with increasing doses of beta-gal-carrying vector and/or upon boosting with a heterologous beta-gal-carrying virus. Most importantly, we found that the strength of the detected immune response against this foreign Ag strongly correlated with reduction in the number of lung metastases. The results from this mouse model have major implications for the implementation of tumor vaccines in humans.     

Inside Back Cover: The conformation of bovine serum albumin adsorbed to the surface of single all‐dielectric nanoparticles following light‐induced heating (J. Biophotonics 7/2018)

Germanium vs Silicon: All‐dielectric nanoparticles provides the heat resistance for proteins under light‐induced heating. Further details can be found in the article by Andrei A. Krasilin et al. ( e201700322 )

     

The conformation of bovine serum albumin adsorbed to the surface of single all‐dielectric nanoparticles following light‐induced heating

Interaction between nanoparticles and biomolecules leads to the formation of biocompatible or bioadverse complexes. Despite the rapid development of nanotechnologies for biology and medicine, relatively little is known about the structure of such complexes. Here, we report on the changes in conformation of a blood protein (bovine serum albumin) adsorbed on the surface of single all‐dielectric nanoparticles (silicon and germanium) following light‐induced heating to 640 K. This protein is considerably more resistant to heat when adsorbed on the nanoparticle than when in solution or in the solid state. Intriguingly, with germanium nanoparticles this heat resistance is more pronounced than with silicon. These observations will facilitate biocompatible usage of all‐dielectric nanoparticles.      

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

Heteromers of Kv7.2/Kv7.3 subunits constitute the main substrate of the neuronal M-current that limits neuronal hyper-excitability and firing frequency. Calmodulin (CaM) binding is essential for surface expression of Kv7 channels, and disruption of this interaction leads to diseases ranging from mild epilepsy to early onset encephalopathy. In this study, we addressed the impact of a charge neutralizing mutation located at the periphery of helix B (K526N). We found that, CaM binding and surface expression was impaired, although current amplitude was not altered. Currents were reduced at a faster rate after activation of a voltage-dependent phosphatase, suggesting that phosphatidylinositol-4,5-bisphosphate (PIP₂) binding was weaker. In contrast, a charge neutralizing mutation located at the periphery of helix A (R333Q) did not affect CaM binding, but impaired trafficking and led to a reduction in current amplitude. Taken together, these results suggest that disruption of CaM-dependent or CaM-independent trafficking of Kv7.2/Kv7.3 channels can lead to pathology regardless of the consequences on the macroscopic ionic flow through the channel.     

The Easter Egg Weevil (Pachyrhynchus) genome reveals syntenic patterns in Coleoptera across 200 million years of evolution

Patterns of genomic architecture across insects remain largely undocumented or decoupled from a broader phylogenetic context. For instance, it is unknown whether translocation rates differ between insect orders. We address broad scale patterns of genome architecture across Insecta by examining synteny in a phylogenetic framework from open-source insect genomes. To accomplish this, we add a chromosome level genome to a crucial lineage, Coleoptera. Our assembly of the Pachyrhynchus sulphureomaculatus genome is the first chromosome scale genome for the hyperdiverse Phytophaga lineage and currently the largest insect genome assembled to this scale. The genome is significantly larger than those of other weevils, and this increase in size is caused by repetitive elements. Our results also indicate that, among beetles, there are instances of long-lasting (>200 Ma) localization of genes to a particular chromosome with few translocation events. While some chromosomes have a paucity of translocations, intra-chromosomal synteny was almost absent, with gene order thoroughly shuffled along a chromosome. This large amount of reshuffling within chromosomes with few inter-chromosomal events contrasts with patterns seen in mammals in which the chromosomes tend to exchange larger blocks of material more readily. To place our findings in an evolutionary context, we compared syntenic patterns across Insecta in a phylogenetic framework. For the first time, we find that synteny decays at an exponential rate relative to phylogenetic distance. Additionally, there are significant differences in decay rates between insect orders, this pattern was not driven by Lepidoptera alone which has a substantially different rate.     

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca²⁺ and Mn²⁺. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.Anthrax is an infectious disease caused by the encapsulated, spore-forming bacterium Bacillus anthracis. Systemic forms of the disease, such as inhalational anthrax, are characterized by nonspecific early symptoms, rapid progression, and lethality approaching 100% (1). The lethality of inhalational anthrax is high even with antibiotic treatment and is caused by accumulation of secreted anthrax toxin (2), which consists of three proteins as follows: protective antigen (PA),² lethal factor (LF), and edema factor. PA binds to membrane receptors, forms pore complexes, and translocates LF and edema factor into the host cell (3, 4). The PA·LF complex is known as the lethal toxin, a virulence factor with pleiotropic action that facilitates establishment of the B. anthracis infection. LF is a Zn²⁺-dependent metalloprotease related to the thermolysin family that cleaves mitogen-activated protein kinase kinases (5).Although the complete mechanism by which LF causes fatal intoxication is still unclear, inhibition of LF proteolytic activity may be an efficient means of preventing anthrax lethality. A better understanding of the LF catalytic mechanism will facilitate rational design and optimization of LF inhibitors with potential clinical applicability. Recent structural (6, 7), mechanistic (8), and in vivo studies (9, 10) of LF point to a sophisticated catalytic mechanism involving accurate recognition of multiple target substrates.Here we use substrate phage display and stopped-flow fluorimetry kinetics to examine both the substrate specificity and elementary steps of substrate processing by LF. Our data allow us to construct a working model of LF-substrate binding and cleavage.     

Knowledge, use and ecology of golden thistle (Scolymus hispanicus L.) in Central Spain

A mounting body of critical research is raising the credibility of Traditional Knowledge (TK) in scientific studies. These studies have gained credibility because their claims are supported by methods that are repeatable and provide data for quantitative analyses that can be used to assess confidence in the results. The theoretical importance of our study is to test consensus (reliability/replicable) of TK within one ancient culture; the Irulas of the Kodiakkarai Reserve Forest (KRF), India. We calculated relative frequency (RF) and consensus factor (F_ic) of TK from 120 Irulas informants knowledgeable of medicinal plants. Our research indicates a high consensus of the Irulas TK concerning medicinal plants. The Irulas revealed a diversity of plants that have medicinal and nutritional utility in their culture and specific ethnotaxa used to treat a variety of illnesses and promote general good health in their communities. Throughout history aboriginal people have been the custodians of bio-diversity and have sustained healthy life-styles in an environmentally sustainable manner. However this knowledge has not been transferred to modern society. We suggest this may be due to the asymmetry between scientific and TK, which demands a new approach that considers the assemblage of TK and scientific knowledge. A greater understanding of TK is beginning to emerge based on our research with both the Irulas and Malasars; they believe that a healthy lifestyle is founded on a healthy environment. These aboriginal groups chose to share this knowledge with society-at-large in order to promote a global lifestyle of health and environmental sustainability.