Vern Winston

Protein adaptations in archaeal extremophiles.

Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity.

NaV1.4 mutations cause hypokalaemic periodic paralysis by disrupting IIIS4 movement during recovery

Cations leaking through the voltage sensor of mutant sodium or calcium channels underlie hypokalaemic periodic paralysis. Groome et al. use muscle fibre recordings, voltage clamp, and molecular dynamics, to investigate recently discovered Nav1.4 channel mutations. They identify a novel voltage sensor movement that may explain the muscle pathology.

Detection of Euryarchaeota and Crenarchaeota in an oxic basalt aquifer

Groundwater from an oxic, fractured basalt aquifer was examined for the presence of Archaea. DNA was extracted from cells concentrated from groundwater collected from five wells penetrating the eastern Snake River Plain Aquifer (Idaho, USA). Polymerase chain reaction (PCR) amplification of 16S rDNA was performed with Archaea-specific primers using both nested (ca. 200-bp product) and direct (ca. 600-bp product) PCR approaches. Estimates of the archaeal diversity were made by separating PCR products from all five wells by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis of partial 16S rDNA sequences from two wells was performed following cloning procedures. Archaea were detected in all wells and the number of DGGE bands per well ranged from two to nine and varied according to PCR approach. There were 30 unique clonal 16S rDNA partial sequences (ca. 600 bp) within a total of 100 clones that were screened from two wells. Twenty-two of the 16S rDNA fragments recovered from the aquifer were related to the Crenarchaeota and Euryarchaeota kingdoms (one large clade of clones in the former and six smaller clades in the latter), with sequences ranging from 23.7 to 95.4% similar to those found in other investigations. The presence of potentially thermophilic or methanogenic Archaea in this fully oxic aquifer may be related to deep thermal sources or elevated dissolved methane concentrations. Many sequences were similar to those that represent non-thermophilic Crenarchaeota of which there are no known cultured members and therefore no putative function.

Characterization of a new bacteriophage which infects bacteria of the genus Acidiphilium.

A novel bacteriophage, termed phi AC1, that infects strains of the genus Acidiphilium (acidophilic, heterotrophic, aerobic, Gram-negative eubacteria) most commonly isolated from acidic mine drainage environments, has been discovered and several of its properties have been determined. This is the first report of a bacteriophage infecting such cells. The virion has a lambdoid morphology and is larger than lambda, as shown by electron microscopy and sucrose gradient centrifugation. The sedimentation coefficient of the virion is approximately 615S. The nucleic acid of phi Ac1 is dsDNA, approximately 102 kb in length. Several experimental results show that phi Ac1 is a temperate phage. The plaques are turbid, and most cells isolated from plaques produced on sensitive cells by filter-sterilized phage preparations contain the phage and are resistant to further phage infection. Southern blot analysis shows that phi Ac1 prophage DNA is integrated into the bacterial genome during the temperature growth phase.

Positively selected sites on the surface glycoprotein (G) of infectious hematopoietic necrosis virus

Mutations in the surface glycoprotein (G) of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that causes significant losses in hatcheries raising salmonid fish, were studied. A 303 nt segment (mid-G region) of this protein from 88 Idaho isolates of IHNV was sequenced. Evidence of positive selection at individual codon sites was estimated by using a Bayesian method (MrBayes). A software algorithm (CPHmodels) was used to construct a three-dimensional (3D) representation of the IHNV protein. The software identified structural homologies between the IHNV G protein and the surface glycoprotein of vesicular stomatitis virus (VSV) and used the VSV structure as a template for predicting the IHNV structure. The amino acids predicted to be under positive selection were mapped onto the proposed IHNV 3D structure and appeared at sites on the surface of the protein where antigen-antibody interaction should be possible. The sites identified as being under positive selection on the IHNV protein corresponded to those reported by others as active sites of mutation for IHNV, and also as antigenic sites on VSV. Knowledge of the sites where genetic variation is positively selected enables a better understanding of the interaction of the virus with its host, and with the host immune system. This information could be used to develop strategies for vaccine development for IHNV, as well as for other viruses.

S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

Glycated hemoglobin is not an accurate indicator of glycemia in rainbow trout.

Glycation occurs when glucose reacts non-enzymatically with proteins. This reaction depends upon time, ambient glucose concentration, and the molecular conformation of reactive amino acids. Little is known about protein glycation in fishes and the main objective of this study was to measure glycated hemoglobin (GHb) in rainbow trout, a glucose-intolerant species, under normoglycemic and hyperglycemic conditions. We also identified GHb isoforms in vivo and analyzed the structural environment surrounding potential glycation sites. Despite similar glycemia to healthy humans, GHb was an order of magnitude lower in rainbow trout (0.6%) compared with humans (6%) and was not affected by long-term hyperglycemia. Species differences in GHb appear to be related to differences in erythrocyte glucose, and differential expression and glycation of hemoglobin (Hb) isoforms may explain intraspecific differences in rainbow trout GHb. Computer analysis of glucose isomers (ringed-open and α- and β-pyranoses) interacting with the β-chain of rainbow trout HbI and HbIV, and human HbA did not reveal structural or energetic constraints for glucose binding (the initial step of glycation) for rainbow trout Hbs. Overall, there are significant differences between Hb glycation in humans and rainbow trout, and GHb does not appear to be an accurate indicator of glycemia over time in rainbow trout.

A novel Ile1455Thr variant in the skeletal muscle sodium channel alpha-subunit in a patient with a severe adult-onset proximal myopathy with electrical myotonia and a patient with mild paramyotonia phenotype

In sodium channelopathies, a severe fixed myopathy caused by a dominant mutation is rare. We describe two unrelated patients with a novel variant, p.Ile1455Thr, with phenotypes of paramyotonia in one case and fixed proximal myopathy with latent myotonia in another. In-vitro whole cell patch-clamp studies show that the mutation slows inactivation and accelerates recovery, in line with other paramyotonia variants with destabilized fast inactivation as pathomechanism. Additionally, p.IleI1455 causes a loss-of-function by reduced membrane insertion, right-shift of activation, and slowed kinetics. Molecular dynamics simulations comparing wild type and mutant Nav1.4 showed that threonine substitution hindered D4S4 mobility in response to membrane depolarization, consistent with effects of the mutation on channel inactivation. The fixed myopathy is likely to be associated to gain-of-function leading to sodium accumulation, regional edema, T-tubular swelling and mitochondrial stress. A possible contribution of the loss-of-function features towards myotonia and myopathy is discussed.

Predicting genome terminus sequences of Bacillus cereus -group bacteriophage using next generation sequencing data

BackgroundMost tailed bacteriophages (phages) feature linear dsDNA genomes. Characterizing novel phages requires an understanding of complete genome sequences, including the definition of genome physical ends.ResultWe sequenced 48 Bacillus cereus phage isolates and analyzed Next-generation sequencing (NGS) data to resolve the genome configuration of these novel phages. Most assembled contigs featured reads that mapped to both contig ends and formed circularized contigs. Independent assemblies of 31 nearly identical I48-like Bacillus phage isolates allowed us to observe that the assembly programs tended to produce random cleavage on circularized contigs. However, currently available assemblers were not capable of reporting the underlying phage genome configuration from sequence data. To identify the genome configuration of sequenced phage in silico, a terminus prediction method was developed by means of ‘neighboring coverage ratios’ and ‘read edge frequencies’ from read alignment files. Termini were confirmed by primer walking and supported by phylogenetic inference of large DNA terminase protein sequences.ConclusionsThe Terminus package using phage NGS data along with the contig circularity could efficiently identify the proximal positions of phage genome terminus. Complete phage genome sequences allow a proposed characterization of the potential packaging mechanisms and more precise genome annotation.

Detection of infectious pancreatic necrosis virus using an inhibition enzyme-linked immunosorbent assay.

An inhibition enzyme-linked immunosorbent assay was used to detect infectious pancreatic necrosis (IPN) virus. In this assay the presence of virus was determined by measuring the decrease in titer of a known antiserum after incubation with a sample suspected to contain virus. The titer of the antiserum was measured with an indirect enzyme-linked assay. Compared to the double antibody sandwich method this assay required fewer reagents (only one anti-IPN serum was required). This assay was also sensitive enough to detect virus at levels of 1 X 10(2) TCID 50/ml. of purified virus and was able to detect virus in samples obtained in the field.